Carbohydrate ligands that bind to IgM antibodies against myelin-associated glycoprotein

ABSTRACT

The invention relates to carbohydrate ligands presenting the minimal Human Natural Killer-1 (HNK-1) epitope that bind to anti-MAG (myelin-associated glycoprotein) IgM antibodies, and their use in diagnosis as well as for the treatment of anti-MAG neuropathy. In particular, the invention relates to disaccharides of formula (I) and (II) wherein Z is optionally substituted phenyl, heteroaryl, arylcarbonyl, or heteroarylmethyl, and to therapeutically acceptable polymers comprising a multitude of substituents of formula (I) and/or formula (II), wherein Z is a bifunctional linker connecting the disaccharides to the polymer backbone.

FIELD OF THE INVENTION

The invention relates to carbohydrate ligands that bind to IgMantibodies against myelin-associated glycoprotein (MAG), polymerscomprising these, and to their use in diagnosis and therapy of anti-MAGneuropathy.

BACKGROUND OF THE INVENTION

Anti-myelin-associated glycoprotein neuropathy is a demyelinatingperipheral neuropathy, caused by autoantibodies recognizing theantigenic HNK-1 carbohydrate epitope, found on myelin-associatedglycoprotein (MAG) and other glycoconjugates of the peripheral nervoussystem (PNS). The clinical picture is characterized by a slowlyprogressing demyelinating, predominantly sensory neuropathy. Thecorrelation of high levels of antibodies and demyelination is wellestablished. Thus, pathological studies on nerve biopsies from patientsshow demyelination and widening of myelin lamellae, as well as depositsof anti-MAG IgM on myelin. Furthermore, therapeutic reduction of the IgMantibody concentration leads to clinical improvement of neuropathicsymptoms. (A. J. Steck et al., Current Opinion in Neurology 2006,19:458-463; M. C. Dalakas, Current Treatment Options in Neurology 2010,12:71-83).

The myelin glycoconjugates that contain the HNK-1 epitope include theglycoproteins MAG, protein zero (P0), peripheral myelin protein-22(PMP22), as well as the glycolipids sulfoglucuronyl paragloboside (SGPG)and sulfoglucuronyl lactosaminyl paragloboside (SGLPG). Severalobservations suggest MAG as major target for the IgM antibodies: (i)Deposits of patients' antibodies to PNS sites are co-localized with MAG,(ii) MAG is selectively lost from myelin, and (iii) the human nervepathology and MAG-knockout mice show characteristic similarities (R. H.Quarles, Journal of Neurochemistry 2007, 100: 1431-1448).

MAG belongs to the family of sialic acid-binding immunoglobulin-likelectins (Siglecs). It is located mainly in periaxonal membranes ofoligodendroglial cells in the CNS and Schwann cells in the PNS and isinvolved in adhesion and signaling processes at the axon-glia interface(R. H. Quarles, 2007, loc. cit.). MAG is strongly glycosylated, i.e. 30%of its molecular weight is contributed by heterogeneous N-linkedoligosaccharides. All of the potential eight N-glycosylation sites ofMAG can carry the HNK-1 epitope. The two glycolipids (SGPG, SGLPG)carrying the HNK-1 epitope contain 3-O-sulfoglucuronic acid (SO₃-3GlcA)as a specific hallmark (T. Ariga et al., J Biol Chem 1987, 262:848-853).Interestingly, the HNK-1 epitope structure of bovine glycoprotein POalso contains this characteristic feature. The similarity between thethree elucidated structures is restricted to the terminal trisaccharide.Consequently the HNK-1 epitope was defined asSO₃-3-GlcA(β1-3)Gal(β1-4)GlcNAc—OH.

The precise carbohydrate epitope recognized by IgM antibodies remainsunclear. A study with SGPG derivatives showed that the IgM antibodiesplace different importance on the carboxyl and the sulfate group.Whereas “intact” SGPG, containing both negatively charged groups, wasreported as optimal epitope for antibody binding (A. A. Ilyas et al., JNeurochemistry 1990, 55:594-601), other studies emphasize the importanceof the length of the carbohydrate chain for antibody recognition.Furthermore, the SO₃-3-GlcA(β1-3)Gal disaccharide epitope seems to bethe minimum requirement for binding (A. Tokuda et al., J. CarbohydrateChemistry 1998, 17:535-546).

SUMMARY OF THE INVENTION

The invention relates to carbohydrate ligands that bind to anti-MAG IgMantibodies, and their use in diagnosis as well as for the treatment ofanti-MAG neuropathy.

In particular the invention relates to disaccharides of formula (I)

and of formula (II)

wherein Z is optionally substituted phenyl, heteroaryl, arylcarbonyl, orheteroarylmethyl.

Furthermore the invention relates to therapeutically acceptable polymerscomprising a multitude of substituents of formula (I) and/or formula(II), wherein Z is a linker connecting said substituent to the polymerbackbone.

The invention relates also to pharmaceutical compositions comprisingthese compounds, diagnostic kits containing these, and to the use ofthese compounds for the diagnosis and therapy of anti-MAG neuropathy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of a competitive binding assay

-   -   (a) Incubation of MAG-coated plates with anti-MAG IgM (patient        sera) and polymer 25. (b) Wash step. (c) Incubation with        anti-human IgM antibody coupled to horseradish peroxidase. (d)        Wash step. (e) Addition of tetramethylbenzidin (TMB)        substrate. (f) Addition of acidic stop solution and measurement        of the optical density.

FIG. 2. Binding curves for compounds 1, 2 and 25

-   -   2(a) The MAG-coated wells were co-incubated with compound 1 (50        mM highest concentration) and the four patient sera MK, DP, KH        and SJ (% ab=% IgM antibody binding to MAG).    -   2(b) Co-incubation of MAG-coated wells with compound 2 (50 mM        highest concentration) together with patient sera MK and SJ.    -   2(c) Co-incubation with compound 25 (15 μM highest        concentration) together with patient sera MK, KH and SJ.        Compound 25 is a polylysine polymer with a defined percentage of        lysine residues coupled to the minimal HNK-1 epitope (1). The        general abbreviation used is as follows: PL(minHNK-1)_(x) with x        defining the percentage of epitope loading in %. In this case        the polymer is PL(minHNK-1)₄₄.    -   2(d) Co-incubation with patient serum KH together with the        polymers PL(minHNK-1)_(x) with x being 10, 25, 31 and 44% (0.5        mM highest concentration).    -   2(e) Co-incubation with the mouse monoclonal anti-HNK-1 IgM        antibody, a positive control antibody, together with the        polymers PL(minHNK-1)_(x) with x being 10, 25, 31 and 44% (0.5        mM highest concentration).

DETAILED DESCRIPTION OF THE INVENTION

A minimal HNK-1 carbohydrate epitope still reliably recognized byanti-MAG IgM antibodies was identified and corresponding disaccharidesprepared both in a sulfated (formula I) and non-sulfated form (formulaII).

The invention relates to these disaccharides of formula (I)

and of formula (II)

wherein Z is optionally substituted phenyl, heteroaryl, arylcarbonyl, orheteroarylmethyl. The sulfate moiety in formula (I) is located inposition 3 of glucuronic acid.

Furthermore the invention relates to polymers comprising a multitude ofsubstituents of formula (I) and/or formula (II), wherein Z is a linkerconnecting said substituent to the polymer backbone.

In particular, linker Z is (bifunctional) aryl, heteroaryl, aryl-loweralkyl, arylcarbonyl, or heteroarylmethyl, wherein aryl or heteroaryl issubstituted by alkylene with 3 to 25 carbon atoms connecting to thepolymer wherein optionally

-   -   (a) one or more carbon atoms of alkylene are replaced by        nitrogen carrying a hydrogen atom, and one of the adjacent        carbon atoms is substituted by oxo, representing an amide        function —NH—CO—; and/or    -   (b) one or more carbon atoms of alkylene are replaced by oxygen;    -   (c) one or more carbon atoms of alkylene are replaced by        sulphur; and/or    -   (d1) the terminal carbon atom connecting to the polymer is        substituted by oxo; or    -   (d2) the terminal carbon atom connecting to the polymer is        replaced by —NH—.

The polymer comprising the multitude of substituents of formula (I)and/or formula (II), wherein Z is a linker connecting said substituentto the polymer backbone, is preferably an α-amino acid polymer, anacrylic acid or methacrylic acid polymer or copolymer, or aN-vinyl-2-pyrrolidone-vinylalcohol copolymer.

Particular examples of polymers of the invention are

-   -   (A) a poly-α-amino acid, wherein the amino acid carries a side        chain aminoalkyl function, such as in poly-lysine, in particular        poly-L-lysine or poly-D-lysine, and the amino group is connected        to a terminal carbonyl group of bifunctional linker Z;    -   (B) a poly-α-amino acid, wherein the amino acid carries a side        chain carbonylalkyl function, such as in poly-aspartic acid or        poly-glutamic acid, and the carbonyl group (which corresponds to        the original carboxy group in aspartic acid and glutamic acid,        respectively) is connected to a terminal —CH₂-group of        bifunctional linker Z;    -   (C) poly-acrylic acid, poly-methacrylic acid or a copolymer of        acrylic and methacrylic acid, wherein the carboxy group is        amidated by a terminal amino group of bifunctional linker Z; and    -   (D) a copolymer of N-vinyl-2-pyrrolidone and vinyl alcohol,        wherein the hydroxy group of the vinyl alcohol part of the        copolymer is connected to a terminal carbonyl group of        bifunctional linker Z.

In a particular embodiment, a polymer (A) comprises the partial formula(III)

wherein

-   -   R¹ is an aminoalkyl substituent connected to linker Z, wherein        the alkylene group of Z carries an oxo group in the terminal        position connected to the amino group of R¹,    -   R² is 2,3-dihydroxypropylthioacetyl-aminoalkyl,    -   and the relation between the two bracketed entities with R¹ and        R², respectively, in the polymer indicates the relation of        disaccharide loading to capped amino function.

For example, R¹ is of formula (IIIa)

and R² is of formula (IIIb)

wherein o is between 1 and 6, preferably 3 or 4, p is between 1 and 6,preferably between 2 and 4, in particular 3, and q is between 1 and 6,preferably between 1 and 4, in particular 2.

When o is 3, substituent R¹ represents a side chain of poly-ornithine,and when o is 4, substituent R¹ represents a side chain of poly-lysine,connected to a linker Z carrying a disaccharide of formula (I) or (II)at the free valence, and R² is2,3-dihydroxy-propylthioacetyl-aminoalkyl, i.e. a capped amino functionhaving a solubilizing substituent.

The poly-amino acid can be linear, hyperbranched or dendritic, asdescribed by Z. Kadlecova et al., Biomacromolecules 2012, 13:3127-3137,for poly-lysine as follows:

The poly-lysine used to prepare polymer (A) of formula (III) haspreferably a molecular weight between 15,000 and 300,000, in particular30,000 to 70,000, and such polymers further connected via a linker Z tocompounds of formula (I) and/or (II) and with a capping2,3-dihydroxypropylthioacetyl residue are preferred.

In a particular embodiment, a polymer (B) comprises the partial formula(III)

wherein

-   -   R¹ is a carbonylalkyl substituent connected to linker Z, wherein        the alkylene group of Z carries a —CH₂-group in the terminal        position connected to the carbonyl group of R¹,    -   R² is 2,3-dihydroxypropylthioacetyl-carbonylalkyl,    -   and the relation between the two bracketed entities with R¹ and        R², respectively, in the polymer indicates the relation of        disaccharide loading to capped carbonyl or carboxy function.

For example, R¹ is of formula (IIIc)

and R² is of formula (IIId)

wherein o is between 1 and 6, preferably 1 or 2, p is between 1 and 6,preferably between 2 and 4, in particular 3, and q is between 1 and 6,preferably between 1 and 4, in particular 2.

When o is 1, substituent R¹ represents a side chain of poly-glutamicacid, and when o is 2, substituent R¹ represents a side chain ofpoly-aspartic acid, connected to a linker Z carrying a disaccharide offormula (I) or (II) at the free valence, and R² is2,3-dihydroxy-propylthioacetyl-carbonylalkyl, i.e. a capped carboxyfunction having a solubilizing substituent.

The poly-aspartic acid used to prepare polymer (B) of formula (III) haspreferably a molecular weight between 15,000 and 300,000, in particular30,000 to 70,000, and such polymers further reacted with linker Zconnected to compounds of formula (I) and/or (II) and with a capping2,3-dihydroxypropylthioalkyl residue are preferred.

In a particular embodiment, a polymer (C) comprises the partial formula(IV)

wherein

-   -   R¹ is a linker Z, wherein the alkylene group of Z carries a        —NH₂— group in the terminal position connected to the carbonyl        group in (IV),    -   R² is 2,3-dihydroxypropylthioacetylaminoalkylamino or a related        amino substituent, and    -   R³ is hydrogen or methyl;    -   and the relation between the two bracketed entities with R¹ and        R², respectively, in the polymer indicates the relation of        disaccharide loading to capped carboxy function.

For example, R¹ is of formula (IVa)

and R² is of formula (IVb)

wherein in q is between 1 and 6, preferably between 4 and 6, and r isbetween 1 and 6, preferably between 1 and 4, in particular 2.

In another embodiment R¹ is of formula (IVd)

and R² is of formula (IVe)

wherein p is between 1 and 10, preferably between 1 and 4, q is between1 and 6, preferably between 4 and 6, and r is between 1 and 6,preferably between 1 and 4, in particular 2.

In another embodiment R¹ is of formula (IVf)

wherein r is between 1 and 6, preferably between 1 and 4, in particular2, and R² is of formula (IVc) (above).

The poly-acrylic acid used to prepare polymer (C) of formula (IV) haspreferably a molecular weight between 30,000 and 400,000, in particular30,000 to 160,000, and such polymers further reacted with linker Zconnected to compounds of formula (I) and/or (II) and with a capping2,3-dihydroxypropylthioacetyl residue are preferred.

In a particular embodiment, a polymer (D) comprises the partial formula(V)

wherein

-   -   R¹ is a linker Z, wherein the alkylene group of Z carries a        aminocarbonyl group in the terminal position connected to the        hydroxyl group in (V),    -   R² is 2,3-dihydroxypropylthioacetylaminoalkylaminocarbonyl or a        related aminocarbonyl substituent,    -   and the relation between the two bracketed entities with R¹ and        R², respectively, in the polymer indicates the relation of        disaccharide loading to capped hydroxy function.

For example, R¹ is of formula (Va)

and R² is of formula (Vb)

wherein q is between 1 and 6, preferably between 4 and 6, and r isbetween 1 and 6, preferably between 1 and 4, in particular 2.

In another embodiment R¹ is of formula (Vc)

and R² is of formula (Vd)

wherein p is between 1 and 10, preferably between 1 and 4, q is between1 and 6, preferably between 4 and 6, and r is between 1 and 6,preferably between 1 and 4, in particular 2.

In another embodiment R¹ is of formula (Ve)

and R² is of formula (Vf)

wherein r is between 1 and 6, preferably between 1 and 4, in particular2.

The copolymer used to prepare polymer (D) of formula (V) has preferablya molecular weight between 30,000 and 400,000, in particular 30,000 to160,000, and such polymers further reacted with linker Z connected tocompounds of formula (I) and/or (II) and with a capping2,3-dihydroxypropylthioacetyl residue are preferred.

The general terms used hereinbefore and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise indicated:

The prefix “lower” denotes a radical having up to and including amaximum of 7, especially up to and including a maximum of 4 carbonatoms, the radicals in question being either linear or branched withsingle or multiple branching.

Where the plural form is used for compounds, salts, and the like, thisis taken to mean also a single compound, salt, or the like.

Double bonds in principle can have E- or Z-configuration. The compoundsof this invention may therefore exist as isomeric mixtures or singleisomers. If not specified both isomeric forms are intended.

Any asymmetric carbon atoms may be present in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration. Thecompounds may thus be present as mixtures of isomers or as pure isomers,preferably as enantiomer-pure diastereomers.

Alkyl (or bifunctional alkylene in a linker) has from 1 to 25, forexample 1 to 12, preferably from 1 to 7 carbon atoms, and is linear orbranched. Alkyl is preferably lower alkyl.

Preferably, (bifunctional) alkylene has from 3 to 25, preferably from 4to 12 carbon atoms.

Lower alkyl has 1 to 7, preferably 1 to 4 carbon atoms and is butyl,such as n-butyl, sec-butyl, isobutyl, tert-butyl, propyl, such asn-propyl or isopropyl, ethyl or methyl. Preferably lower alkyl is methylor ethyl.

Cycloalkyl has preferably 3 to 7 ring carbon atoms, and may beunsubstituted or substituted, e.g. by lower alkyl or lower alkoxy.Cycloalkyl is, for example, cyclohexyl, cyclopentyl, methylcyclopentyl,or cyclopropyl, in particular cyclopropyl.

Aryl stands for a mono- or bicyclic fused ring aromatic group with 5 to10 carbon atoms optionally carrying substituents, such as phenyl,1-naphthyl or 2-naphthyl, or also a partially saturated bicyclic fusedring comprising a phenyl group, such as indanyl, indolinyl, dihydro- ortetrahydronaphthyl, all optionally substituted. Preferably, aryl isphenyl, indanyl, indolinyl or tetrahydronaphthyl, in particular phenyl.

The term “aryl carrying substituents” stands for aryl substituted by upto four substituents independently selected from lower alkyl, halo-loweralkyl, cycloalkyl-lower alkyl, carboxy-lower alkyl, loweralkoxycarbonyl-lower alkyl; arylalkyl or heteroarylalkyl, wherein arylor heteroaryl are unsubstituted or substituted by up to threesubstituents selected from lower alkyl, cyclopropyl, halo-lower alkyl,lower alkoxy, hydroxysulfonyl, aminosulfonyl, tetrazolyl, carboxy,halogen, amino, cyano and nitro; hydroxy-lower alkyl, lower alkoxy-loweralkyl, aryloxy-lower alkyl, heteroaryloxy-lower alkyl, aryl-loweralkoxy-lower alkyl, heteroaryl-lower alkoxy-lower alkyl, loweralkoxy-lower alkoxy-lower alkyl; aminoalkyl wherein amino isunsubstituted or substituted by one or two substituents selected fromlower alkyl, hydroxy-lower alkyl, alkoxy-lower alkyl and amino-loweralkyl, or by one substituent alkylcarbonyl or mercaptoalkylcarbonyl,alkoxycarbonyl, amino-lower alkoxycarbonyl, lower alkoxy-loweralkoxycarbonyl and aminocarbonyl, or wherein the two substituents onnitrogen form together with the nitrogen heterocyclyl; optionallysubstituted alkenyl, optionally substituted alkinyl, cycloalkyl, aryl,heteroaryl, heterocyclyl, hydroxy, lower alkoxy, halo-lower alkoxy,lower alkoxy-lower alkoxy, cycloalkyl-lower alkoxy, aryloxy, aryl-loweralkoxy, aryloxy-lower alkoxy, heteroaryloxy, heteroaryl-lower alkoxy,heteroaryloxy-lower alkoxy, optionally substituted alkenyloxy,optionally substituted alkinyloxy, cycloalkyloxy, heterocyclyloxy,hydroxysulfonyloxy; alkyl mercapto, hydroxysulfinyl, alkylsulfinyl,halo-lower alkylsulfinyl, hydroxysulfonyl, alkylsulfonyl, arylsulfonyl,heteroarylsulfonyl; aminosulfonyl wherein amino is unsubstituted orsubstituted by one or two substituents selected from lower alkyl,cycloalkyl-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl,cycloalkyl, optionally substituted phenyl, optionally substitutedphenyl-lower alkyl, optionally substituted heteroaryl and optionallysubstituted heteroaryl-lower alkyl, or wherein the two substituents onnitrogen form together with the nitrogen heterocyclyl; amino optionallysubstituted by one or two substituents selected from lower alkyl,cycloalkyl-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl,di-lower alkylamino-lower alkyl, cycloalkyl, optionally substitutedphenyl-lower alkyl and optionally substituted heteroaryl-lower alkyl, orby one substituent optionally substituted phenyl, optionally substitutedheteroaryl, alkylcarbonyl, optionally substituted phenylcarbonyl,optionally substituted pyridylcarbonyl, alkoxycarbonyl or aminocarbonyl,and wherein alkyl or lower alkyl in each case may be substituted byhalogen, lower alkoxy, aryl, heteroaryl or optionally substituted amino,or wherein the two substituents on nitrogen form together with thenitrogen heterocyclyl; carboxymethylamino or loweralkoxycarbonylmethylamino substituted at the methyl group such that theresulting substituent corresponds to one of the 20 naturally occurringstandard amino acids, aminomethylcarbonylamino substituted at the methylgroup such that the resulting acyl group corresponds to one of the 20naturally occurring standard amino acids; lower alkylcarbonyl,halo-lower alkylcarbonyl, optionally substituted phenylcarbonyl,optionally substituted heteroarylcarbonyl, carboxy, loweralkoxycarbonyl, lower alkoxy-lower alkoxycarbonyl; aminocarbonyl whereinamino is unsubstituted or substituted by one hydroxy or amino group orone or two substituents selected from lower alkyl, cycloalkyl-loweralkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, cycloalkyl,optionally substituted phenyl-lower alkyl and optionally substitutedheteroaryl-lower alkyl, or wherein the two substituents on nitrogen formtogether with the nitrogen heterocyclyl; cyano, halogen, and nitro; andwherein two substituents in ortho-position to each other can form a 5-,6- or 7-membered carbocyclic or heterocyclic ring containing one, two orthree oxygen atoms, one or two nitrogen atoms and/or one sulfur atom,wherein the nitrogen atoms are optionally substituted by lower alkyl,lower alkoxy-lower alkyl or lower alkylcarbonyl.

In particular, the substituents may be independently selected from loweralkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl,optionally substituted alkenyl, optionally substituted alkinyl,cyclohexyl, cyclopropyl, aryl, heteroaryl, heterocyclyl, hydroxy, loweralkoxy, halo-lower alkoxy, lower alkoxy-lower alkoxy, cycloalkyloxy,phenoxy, hydroxysulfonyloxy; alkylmercapto, hydroxysulfinyl,alkylsulfinyl, halo-lower alkylsulfinyl, hydroxysulfonyl, alkylsulfonyl,arylsulfonyl, heteroarylsulfonyl; aminosulfonyl wherein amino isunsubstituted or substituted by one or two substituents selected fromlower alkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl, loweralkoxy-lower alkyl and optionally substituted phenyl-lower alkyl, orwherein the two substituents on nitrogen form together with the nitrogenheterocyclyl; amino optionally substituted by one or two substituentsselected from lower alkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl,lower alkoxy-lower alkyl, di-lower alkylamino-lower alkyl, cycloalkyl,or by one substituent optionally substituted phenyl, optionallysubstituted heteroaryl, alkylcarbonyl, optionally substitutedphenylcarbonyl, optionally substituted pyridylcarbonyl, alkoxycarbonylor aminocarbonyl, or wherein the two substituents on nitrogen formtogether with the nitrogen heterocyclyl; carboxymethylamino or loweralkoxycarbonylmethylamino substituted at the methyl group such that theresulting substituent corresponds to one of the 20 naturally occurringstandard amino acids, aminomethylcarbonylamino substituted at the methylgroup such that the resulting acyl group corresponds to one of the 20naturally occurring standard amino acids; lower alkylcarbonyl,halo-lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, loweralkoxy-lower alkoxycarbonyl; aminocarbonyl wherein amino isunsubstituted or substituted by one hydroxy or amino group or one or twosubstituents selected from lower alkyl, hydroxy-lower alkyl, loweralkoxy-lower alkyl, optionally substituted phenyl-lower alkyl andoptionally substituted heteroaryl-lower alkyl, or wherein the twosubstituents on nitrogen form together with the nitrogen heterocyclyl;cyano, halogen, and nitro; and wherein two substituents inortho-position to each other can form a 5- or 6-membered heterocyclicring containing one or two oxygen atoms and/or one or two nitrogenatoms, wherein the nitrogen atoms are optionally substituted by loweralkyl, lower alkoxy-lower alkyl or lower alkylcarbonyl.

In optionally substituted phenyl, substituents are preferably loweralkyl, halo-lower alkyl, lower alkoxy-lower alkyl, amino-lower alkyl,acylamino-lower alkyl, cyclopropyl, hydroxy, lower alkoxy, halo-loweralkoxy, lower alkoxy-lower alkoxy, methylenedioxy, hydroxy-sulfonyloxy,carboxy, lower alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl,tetrazolyl, hydroxysulfonyl, aminosulfonyl, halo, cyano or nitro, inparticular lower alkoxy, amino-lower alkyl, acylamino-lower alkyl,carboxy, lower alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl,tetrazolyl, or aminosulfonyl.

Heteroaryl represents an aromatic group containing at least oneheteroatom selected from nitrogen, oxygen and sulfur, and is mono- orbicyclic, optionally carrying substituents. Monocyclic heteroarylincludes 5 or 6 membered heteroaryl groups containing 1, 2, 3 or 4heteroatoms selected from nitrogen, sulfur and oxygen. Bicyclicheteroaryl includes 9 or 10 membered fused-ring heteroaryl groups.Examples of heteroaryl include pyrrolyl, thienyl, furyl, pyrazolyl,imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl,thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl,pyrimidinyl, pyrazinyl, and benzo or pyridazo fused derivatives of suchmonocyclic heteroaryl groups, such as indolyl, benzimidazolyl,benzofuryl, quinolinyl, isoquinolinyl, quinazolinyl, pyrrolopyridine,imidazopyridine, or purinyl, all optionally substituted. Preferably,heteroaryl is pyridyl, pyrimdinyl, pyrazinyl, pyridazinyl, thienyl,pyrazolyl, imidazolyl, thiazolyl, oxadiazolyl, triazolyl, oxazolyl,isoxazolyl, isothiazolyl, pyrrolyl, indolyl, pyrrolopyridine orimidazopyridine; in particular pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, pyrazolyl, imidazolyl, thiazolyl, oxadiazolyl, triazolyl,indolyl, pyrrolopyridine or imidazopyridine.

The term “heteroaryl carrying substituents” stands for heteroarylsubstituted by up to three substituents independently selected fromlower alkyl, halo-lower alkyl, cycloalkyl-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, aryloxy-lower alkyl,heteroaryloxy-lower alkyl, lower alkoxy-lower alkoxy-lower alkyl;aminoalkyl, wherein amino is unsubstituted or substituted by one or twosubstituents selected from lower alkyl, hydroxy-lower alkyl,alkoxy-lower alkyl, amino-lower alkyl, alkylcarbonyl, alkoxycarbonyl,amino-lower alkoxycarbonyl, lower alkoxy-lower alkoxycarbonyl andaminocarbonyl; optionally substituted alkenyl, optionally substitutedalkinyl, cycloalkyl; aryl, heteroaryl, arylalkyl or heteroarylalkyl,wherein aryl or heteroaryl are unsubstituted or substituted by up tothree substituents selected from lower alkyl, halo-lower alkyl, loweralkoxy, halogen, amino, cyano and nitro; hydroxy, lower alkoxy,halo-lower alkoxy, lower alkoxy-lower alkoxy, cycloalkyloxy,cycloalkyl-lower alkoxy, aryloxy, aryl-lower alkoxy, heteroaryloxy,heteroaryl-lower alkoxy, alkenyloxy, alkinyloxy, alkylmercapto,alkylsulfinyl, halo-lower alkylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroarylsulfonyl, aminosulfonyl wherein amino is unsubstituted orsubstituted by one or two substituents selected from lower alkyl,cycloalkyl-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl,cycloalkyl, optionally substituted phenyl, optionally substitutedphenyl-lower alkyl, optionally substituted heteroaryl and optionallysubstituted heteroaryl-lower alkyl, or wherein the two substituents onnitrogen form together with the nitrogen heterocyclyl; amino optionallysubstituted by one or two substituents selected from lower alkyl,cycloalkyl-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl,di-lower alkylamino-lower alkyl, cycloalkyl, optionally substitutedphenyl, optionally substituted phenyl-lower alkyl, optionallysubstituted heteroaryl, optionally substituted heteroaryl-lower alkyl,alkylcarbonyl, alkoxycarbonyl or aminocarbonyl, and wherein alkyl orlower alkyl in each case may be substituted by halogen, lower alkoxy,aryl, heteroaryl or optionally substituted amino, or wherein the twosubstituents on nitrogen form together with the nitrogen heterocyclyl;lower alkylcarbonyl, halo-lower alkylcarbonyl, optionally substitutedphenylcarbonyl, carboxy, lower alkoxycarbonyl, lower alkoxy-loweralkoxycarbonyl; aminocarbonyl wherein amino is unsubstituted orsubstituted by one hydroxy or amino group or one or two substituentsselected from lower alkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl,lower alkoxy-lower alkyl, cycloalkyl, optionally substituted phenyl,optionally substituted phenyl-lower alkyl, optionally substitutedheteroaryl and optionally substituted heteroaryl-lower alkyl, or whereinthe two substituents on nitrogen form together with the nitrogenheterocyclyl; cyano, halogen, and nitro.

In particular, the substituents on heteroaryl may be independentlyselected from lower alkyl, halo-lower alkyl, cycloalkyl-lower alkyl,lower alkoxy-lower alkyl, lower alkoxy-lower alkoxy-lower alkyl,optionally substituted alkenyl, optionally substituted alkinyl,cycloalkyl, aryl, heteroaryl, hydroxy, lower alkoxy, cycloalkyloxy,alkenyloxy, alkinyloxy, alkyl-mercapto, alkylsulfinyl, halo-loweralkylsulfinyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl wherein aminois unsubstituted or substituted by one or two substituents selected fromlower alkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl, loweralkoxy-lower alkyl, cycloalkyl, optionally substituted phenyl,optionally substituted phenyl-lower alkyl, optionally substitutedheteroaryl and optionally substituted heteroaryl-lower alkyl, or whereinthe two substituents on nitrogen form together with the nitrogenheterocyclyl; amino optionally substituted by one or two substituentsselected from lower alkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl,lower alkoxy-lower alkyl, di-lower alkylamino-lower alkyl, cycloalkyl,alkylcarbonyl, alkoxycarbonyl or aminocarbonyl, and wherein alkyl orlower alkyl in each case may be substituted by lower alkoxy oroptionally substituted amino, or wherein the two substituents onnitrogen form together with the nitrogen heterocyclyl; loweralkyl-carbonyl, halo-lower alkylcarbonyl, carboxy, lower alkoxycarbonyl,lower alkoxy-lower alkoxycarbonyl; aminocarbonyl wherein amino isunsubstituted or substituted by one hydroxy or amino group or one or twosubstituents selected from lower alkyl, cycloalkyl-lower alkyl,hydroxy-lower alkyl, lower alkoxy-lower alkyl or cycloalkyl, or whereinthe two substituents on nitrogen form together with the nitrogenheterocyclyl; cyano, halogen, and nitro.

In optionally substituted heteroaryl, substituents are preferably loweralkyl, halo-lower alkyl, lower alkoxy-lower alkyl, hydroxy, loweralkoxy, halo-lower alkoxy, lower alkoxy-lower alkoxy, methylenedioxy,carboxy, lower alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl,tetrazolyl, aminosulfonyl, halo, cyano or nitro.

Alkenyl contains one or more, e.g. two or three, double bonds, and ispreferably lower alkenyl, such as 1- or 2-butenyl, 1-propenyl, allyl orvinyl.

Alkinyl is preferably lower alkinyl, such as propargyl or acetylenyl.

In optionally substituted alkenyl or alkinyl, substituents arepreferably lower alkyl, lower alkoxy, halo, optionally substituted arylor optionally substituted heteroaryl, and are connected with a saturatedor unsaturated carbon atom of alkenyl or alkinyl.

Heterocyclyl designates preferably a saturated, partially saturated orunsaturated, mono- or bicyclic ring containing 4-10 atoms comprisingone, two or three heteroatoms selected from nitrogen, oxygen and sulfur,which may, unless otherwise specified, be carbon or nitrogen linked,wherein a ring nitrogen atom may optionally be substituted by a groupselected from lower alkyl, amino-lower alkyl, aryl, aryl-lower alkyl andacyl, and a ring carbon atom may be substituted by lower alkyl,amino-lower alkyl, aryl, aryl-lower alkyl, heteroaryl, lower alkoxy,hydroxy or oxo, or which may be fused with an optionally substitutedbenzo ring. Substituents considered for substituted benzo are thosementioned above for optionally substituted aryl. Examples ofheterocyclyl are pyrrolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl,morpholinyl, piperazinyl, dioxolanyl, tetrahydro-furanyl andtetrahydropyranyl, and optionally substituted benzo fused derivatives ofsuch monocyclic heterocyclyl, for example indolinyl, benzoxazolidinyl,benzothiazolidinyl, tetrahydroquinolinyl, and benzodihydrofuryl.

Acyl designates, for example, alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, aryl-lower alkylcarbonyl, or heteroarylcarbonyl. Loweracyl is preferably lower alkylcarbonyl, in particular propionyl oracetyl.

Hydroxyalkyl is especially hydroxy-lower alkyl, preferablyhydroxymethyl, 2-hydroxyethyl or 2-hydroxy-2-propyl.

Cyanoalkyl designates preferably cyanomethyl and cyanoethyl.

Haloalkyl is preferably fluoroalkyl, especially trifluoromethyl,3,3,3-trifluoroethyl or pentafluoroethyl.

Halogen is fluorine, chlorine, bromine, or iodine.

Lower alkoxy is especially methoxy, ethoxy, isopropyloxy, ortert-butyloxy.

Arylalkyl includes aryl and alkyl as defined hereinbefore, and is e.g.benzyl, 1-phenethyl or 2-phenethyl.

Heteroarylalkyl includes heteroaryl and alkyl as defined hereinbefore,and is e.g. 2-, 3- or 4-pyridylmethyl, 1- or 2-pyrrolylmethyl,1-pyrazolylmethyl, 1-imidazolylmethyl, 2-(1-imidazolyl)ethyl or3-(1-imidazolyl)propyl.

In substituted amino, the substituents are preferably those mentioned assubstituents hereinbefore. In particular, substituted amino isalkylamino, dialkylamino, optionally substituted arylamino, optionallysubstituted arylalkylamino, lower alkylcarbonylamino, benzoylamino,pyridylcarbonylamino, lower alkoxycarbonylamino or optionallysubstituted aminocarbonylamino.

Particular salts considered are those replacing the hydrogen atoms ofthe sulfate group and the carboxylic acid function. Suitable cationsare, e.g., sodium, potassium, calcium, magnesium or ammonium cations, oralso cations derived by protonation from primary, secondary or tertiaryamines containing, for example, lower alkyl, hydroxy-lower alkyl orhydroxy-lower alkoxy-lower alkyl groups, e.g., 2-hydroxyethylammonium,2-(2-hydroxyethoxy)ethyldimethylammonium, diethylammonium,di(2-hydroxyethyl)ammonium, trimethylammonium, triethylammonium,2-hydroxyethyldimethylammonium, or di(2-hydroxyethyl)methylammonium,also from correspondingly substituted cyclic secondary and tertiaryamines, e.g., N-methylpyrrolidinium, N-methylpiperidinium,N-methyl-morpholinium, N-2-hydroxyethylpyrrolidinium,N-2-hydroxyethylpiperidinium, or N-2-hydroxyethylmorpholinium, and thelike.

In view of the close relationship between the novel compounds in freeform and those in the form of their salts, including those salts thatcan be used as intermediates, for example in the purification oridentification of the novel compounds, any reference to the freecompounds hereinbefore and hereinafter is to be understood as referringalso to the corresponding salts, and vice versa, as appropriate andexpedient.

Preferably Z is unsubstituted or substituted phenyl.

In particular, the invention refers to compounds of formula (I) or (II),wherein Z is optionally substituted phenyl.

Preferred substituents considered for Z with the meaning of thementioned aryl groups, e.g. phenyl, are lower alkyl, halo-lower alkyl,hydroxy-lower alkyl, lower alkoxy-lower alkyl, amino-lower alkyl, loweralkanecarbonylamino-lower alkyl, mercapto-loweralkane-carbonylamino-lower alkyl, optionally substituted alkenyl,optionally substituted alkinyl, cyclohexyl, cyclopropyl, aryl,heteroaryl, heterocyclyl, hydroxy, lower alkoxy, halo-lower alkoxy,lower alkoxy-lower alkoxy, cycloalkyloxy, hydroxysulfonyloxy; mercapto,alkylmercapto, hydroxysulfinyl, alkylsulfinyl, halo-lower alkylsulfinyl,hydroxysulfonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,aminosulfonyl wherein amino is unsubstituted or substituted by one ortwo substituents selected from lower alkyl, cycloalkyl-lower alkyl,hydroxy-lower alkyl, lower alkoxy-lower alkyl, optionally substitutedphenyl-lower alkyl and optionally substituted heteroaryl-lower alkyl, orwherein the two substituents on nitrogen form together with the nitrogenheterocyclyl; amino optionally substituted by one or two substituentsselected from lower alkyl, cycloalkyl-lower alkyl, hydroxy-lower alkyl,lower alkoxy-lower alkyl and di-lower alkylamino-lower alkyl, or by onesubstituent cycloalkyl, optionally substituted phenyl, optionallysubstituted heteroaryl, alkylcarbonyl, optionally substitutedphenylcarbonyl, optionally substituted pyridylcarbonyl, alkoxycarbonylor aminocarbonyl, or wherein the two substituents on nitrogen formtogether with the nitrogen heterocyclyl; carboxymethylamino or loweralkoxycarbonylmethylamino substituted at the methyl group such that theresulting substituent corresponds to one of the 20 naturally occurringstandard amino acids, aminomethylcarbonylamino substituted at the methylgroup such that the resulting acyl group corresponds to one of the 20naturally occurring standard amino acids; lower alkylcarbonyl,halo-lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, loweralkoxy-lower alkoxycarbonyl; aminocarbonyl wherein amino isunsubstituted or substituted by one hydroxy or amino group or one or twosubstituents selected from lower alkyl, hydroxy-lower alkyl, loweralkoxy-lower alkyl, optionally substituted phenyl-lower alkyl andoptionally substituted heteroaryl-lower alkyl, or wherein the twosubstituents on nitrogen form together with the nitrogen heterocyclyl;cyano, halogen, and nitro; and wherein two substituents inortho-position to each other can form a 5- or 6-membered heterocyclicring containing one or two oxygen atoms and/or one or two nitrogenatoms, wherein the nitrogen atoms are optionally substituted by loweralkyl, lower alkoxy-lower alkyl or lower alkylcarbonyl.

Particularly preferred Z is p-methoxyphenyl, 4-(2-aminoethyl)phenyl or4-(2-(4-mercaptobutanoylamino)ethyl)phenyl.

In polymers comprising a multitude of substituents of formula (I) and/orformula (II), a particular linker Z is (bifunctional) aryl, heteroaryl,aryl-lower alkyl, arylcarbonyl, or heteroarylmethyl, wherein aryl orheteroaryl is substituted by —(CH₂)₂NH(C═O)(CH₂)₃S—CH₂—(C═O)— connectingto the polymer with aminoalkyl side chains at the C═O function.

More particularly linker Z is phenyl substituted by—(CH₂)₂NH(C═O)(CH₂)₃S—CH₂—(C═O)— connecting to the polymer withaminoalkyl side chains at the C═O function.

A preferred polymer in polymers comprising a multitude of substituentsof formula (I) and/or formula (II) is polylysine, in particularpoly-L-lysine.

Preferably the molecular weight of the polylysine is 1,000 to 300,000kD, preferably 10,000 to 100,000 kD. Particularly preferred is amolecular weight of approximately 50,000 kD, 125,000 kD or 200,000 kD.Most preferred is a molecular weight of approximately 50,000 kD.

In particular the invention relates to such polymers wherein therelative loading of polymer backbone with the disaccharide of formula(I) and/or (II) is 10-80%, meaning that 10-80% of all lysine side chainsin the polymer are coupled/reacted with a linker carrying adisaccharide, the remaining amino functions being capped. Preferably theloading of the polymer is 30-60%, more preferably 40-50%.

In a particular embodiment, the sulfated minimal HNK-1 epitope 22carrying a linker with a terminal sulfhydryl function was synthesizedand reacted in a substochiometric amount with the activated(chloroacetylated) lysine polymer 24. The carbohydrate loading (40%) wasdetermined by ¹H NMR. The starting polymer 23 had an average molecularweight (MW) of 50 kD, whereas the final polymer (25) with 40% minimalHNK-1 epitope loading had a calculated average MW of 123 kD.

The synthesized carbohydrate monomers (1 and 2) and the polymer 25 weretested in an established ELISA assay (Bühlmann Laboratories,Schonenbuch, Switzerland) applied for the diagnosis of anti-MAGneuropathy and for therapy control in clinic. The assay is used todetermine serum concentration of anti-MAG IgM autoantibodies. The assaywas modified to a competitive binding assay. The synthesized compoundsand serum samples containing anti-MAG IgM antibodies are given into 96well plates, coated with purified MAG from human brain. Immobilized MAGand the synthesized compounds compete for binding to the anti-MAG IgMantibodies. After a washing step MAG-bound IgM antibodies are detectedwith a horseradish peroxidase labeled antibody, followed by acolorimetric reaction. Successful competition of the compounds with MAGleads to a decrease in measured OD_(450 nm) (optical density), becausethey block the binding sites of IgM antibodies, preventing them frombinding to MAG. The principle of the assay is depicted in FIG. 1. Forthe evaluation of the compounds, four sera from different patients (MK,DP, KH, SJ) with reported high anti-MAG IgM antibody titers were chosen.IgM antibody concentrations were determined for each serum inpreliminary experiments. Serum dilutions with measured OD_(450 nm)values around 1.0 were chosen for the assay, to be able to compare themeasured IC₅₀ values (half maximal inhibitory concentration) which areantibody concentration dependent. Serum dilutions: DP 1:2′500, KH1:3,000, SJ 1:7,500, MK 1:23,000. The two sera that served as negativecontrols (dilution 1:1000) showed no binding to MAG.

IC₅₀ values of compound 1 were determined for all sera. Those ofcompound 2 were determined for serum MK with the highest antibodyaffinity for 1 and for serum SJ with the lowest antibody affinity for 1.The results are shown in the Table below. The assay was repeated fourtimes. From the received binding curves for each serum, the three bestfitted were chosen and normalized for IC₅₀ calculation. The bindingcurves are shown in FIG. 2. For curve generation of compound 2 anartificially high concentration point at 500 mM with 0% antibody bindingwas added because even at the highest concentration of 2 (50 mM) theinhibition of antibody binding was not 100%. The IC₅₀ values forcompound 2 are therefore to be considered as approximated values,although they changed only marginally upon addition of the highconcentration point. Under the same assay conditions the carbohydratepolymer was tested with the sera KH, MK and SJ. The measurements wererepeated at least three times. The three best fitted curves for eachserum were chosen for IC₅₀ calculation. The non-normalized bindingcurves are shown in FIG. 2.

TABLE IC₅₀ values of compounds 1, 2 and the minimal HNK-1 polymer (25)for the four patient sera including standard deviations. Compound 1Compound 2 Polymer (25) Serum IC₅₀ (μM) IC₅₀ (mM) IC₅₀ (nM) MK 124.2 ±9.5  29.0 ± 0.5  2.5 ± 0.1 DP 536.1 ± 23.5 n.d. n.d. KH 614.2 ± 20.1n.d. 18.3 ± 2.2 SJ 793.1 ± 24.0 10.0 ± 1.0 14.8 ± 0.6

The data from the biological evaluation of 1 and 2 clearly showdifferent affinities of the IgM antibodies of each serum to thesynthesized disaccharides. Disaccharide 1 shows a superior bindingaffinity towards the IgM antibodies when compared to disaccharide 2,which lacks the sulfate moiety. The sulfate group seems to be essentialto the synthesized minimal HNK-1 epitope for antibody binding.Nevertheless, it is not equally important for all sera. Serum MK showedhigh requirement for the sulfate with an approximately 230-fold weakerbinding to the unsulfated disaccharide. Serum SJ on the other handshowed only 12.6-fold lower binding affinity to the unsulfateddisaccharide. The carboxyl group of GlcA seems to be more important tothis serum.

For all IgM antibodies, the sulfate moiety is required for binding inthe μM range. It is surprising that the sulfated minimal HNK-1 epitopeis capable of inhibiting the antibody binding to MAG in the μMconcentration range. This suggests the possibility that the terminalaromatic moiety of the disaccharide is involved in binding, as ifmimicking the third sugar (GlcNAc) of the HNK-1 epitope. The aromaticring could undergo cation-Tr interaction or π-π stacking.

The causal relationship between anti-MAG autoantibodies and neuropathydevelopment in anti-MAG neuropathy patients is widely accepted today(M.C. Dalakas, Current Treatment Options in Neurology 2010, 12:71-83).The antigenic determinant for these antibodies is the HNK-1 carbohydrateepitope, the trisaccharide SO₄-3-GlcA(β1-3)Gal(β1-4)GlcNAc—OH which isalso recognized by the HNK-1 antibody.

According to the present invention it is shown that carbohydrate ligandsblocking the IgM antibody binding sites prevent the antibody binding toMAG and other myelin targets.

It is shown that disaccharide ligands of formula (I) and (II), minimalHNK-1 carbohydrate epitopes, which are much easier to prepare thanlarger carbohydrates, retain affinity to the IgM antibodies, and areuseful for diagnostic and therapeutic purposes.

Compounds related to substance 1 and 2 are known in the state of theart, but not such compounds containing arylic aglycons. Aromaticresidues Z take part in the binding process to the anti-MAG IgMantibodies and therefore bestow a substantial benefit on compounds suchas (I) and/or (II) with arylic aglycons.

In the case of the sulfated structure (I) an ethylamine substitutedderivative of a pentasaccharide is published (A. V. Kornilov,Carbohydrate Research 2000, 329:717-730). In the case of structure (II)the unsubstituted derivative (R═H) and derivatives with common alkylresidues are published. In addition to the presently claimed arylsubstitution, such as para-methoxyphenyl, the approach to present thisepitope in multiple copies on a suitable polymer is novel.

Natural carbohydrates generally display low binding affinity for theirbinding partners. In biological systems sufficient affinity is oftenachieved by multivalent presentation of carbohydrates, as well asoligovalent presentation of carbohydrate recognizing domains (CRDs) ofcarbohydrate binding proteins (B. Ernst and J. L. Magnani, NatureReviews Drug Discovery 2009, 8:661-677). This is also the case for thebinding of IgM antibodies to MAG: MAG presents up to eight HNK-1epitopes on its extracellular domains.

In a particularly preferred embodiment, the invention relates topolymers comprising a multitude of substituents of formula (I) and/orformula (II), wherein the polymer is poly-L-lysine and Z is abifunctional linker connecting said substituent to the polymer backbone.

Poly-L-lysine is biodegradable and therefore suitable for therapeuticalapplication. The exemplified minimal HNK-1 polymer shows a massiveincrease in binding affinity toward the pathogenic IgM antibodies. Theinhibitory activity, now being in the low nM range, is increased by afactor of at least 34,000 compared to the monomer (serum KH). Theaffinity increase obtained for serum MK and SJ was approximately 50,000(see Table above). These findings clearly indicate the multivalentnature of the antigen-antibody interaction.

The exemplified minimal HNK-1 polymer serves as substitute antigen forpurified human brain MAG currently used in a diagnostic ELISA assay forthe detection of anti-MAG IgM antibodies.

The compounds of the invention have valuable pharmacological properties.The invention also relates to compounds as defined hereinbefore for useas medicaments. A compound according to the invention shows prophylacticand therapeutic efficacy especially against anti-MAG neuropathy.

A compound of formula (I) or (II), or polymers comprising these, can beadministered alone or in combination with one or more other therapeuticagents, possible combination therapy taking the form of fixedcombinations, or the administration of a compound of the invention andone or more other therapeutic agents being staggered or givenindependently of one another, or the combined administration of fixedcombinations and one or more other therapeutic agents.

Therapeutic agents for possible combination are especiallyimmunosuppressive agents. Examples are purine analogues such asfludarabine and/or cladribine, furthermore the chimeric monoclonalantibody rituximab (A. J. Steck et al., Current Opinion in Neurology2006, 19:458-463).

In another particular embodiment, the invention relates to the use ofthe compounds of the invention in a diagnostic assay for anti-MAGneuropathy. In particular, the invention relates to kits comprising thecompounds of formula (I) or (II) as defined above, and also polymers ofthe invention comprising such compounds as substituents.

The present invention relates to a method of diagnosis of anti-MAGneuropathy, wherein the level of IgM against MAG is determined in a bodyfluid sample, e.g. serum, and a high level is indicative of thedevelopment and the severity of anti-MAG neuropathy.

Other body fluids than serum useful for determination of IgM against MAGare, e.g., whole blood, cerebrospinal fluid or extracts from solidtissue.

Any known method may be used for the determination of the level of IgMagainst MAG in body fluids. Methods considered are, e.g., ELISA, RIA,EIA, or microarray analysis.

A preferred method for the determination of IgM against MAG in humanbody fluids, e.g. in serum, is an ELISA. In such an embodiment,microtiter plates are coated with compounds of formula (I) or (II), orpreferably polymers of the invention comprising such compounds assubstituents. The plates are then blocked and the sample or a standardsolution is loaded. After incubation, an anti-IgM antibody is applied,e.g. an anti-IgM antibody directly conjugated with a suitable label,e.g. with an enzyme for chromogenic detection. Alternatively, apolyclonal rabbit (or mouse) anti-IgM antibody is added. A secondantibody detecting the particular type of the anti-IgM antibody, e.g. ananti-rabbit (or anti-mouse) antibody, conjugated with a suitable label,e.g. the enzyme for chromogenic detection as above, is then added.Finally the plate is developed with a substrate for the label in orderto detect and quantify the label, being a measure for the presence andamount of IgM against MAG. If the label is an enzyme for chromogenicdetection, the substrate is a colour-generating substrate of theconjugated enzyme. The colour reaction is then detected in a microplatereader and compared to standards.

It is also possible to use antibody fragments. Suitable labels arechromogenic labels, i.e. enzymes which can be used to convert asubstrate to a detectable colored or fluorescent compound, spectroscopiclabels, e.g. fluorescent labels or labels presenting a visible color,affinity labels which may be developed by a further compound specificfor the label and allowing easy detection and quantification, or anyother label used in standard ELISA.

Other preferred methods of IgM against MAG detection areradioimmunoassay or competitive immunoassay and chemiluminescencedetection on automated commercial analytical robots. Microparticleenhanced fluorescence, fluorescence polarized methodologies, or massspectrometry may also be used. Detection devices, e.g. microarrays, areuseful components as readout systems for IgM against MAG.

In a further embodiment the invention relates to a kit suitable for anassay as described above, in particular an ELISA, comprising compoundsof formula (I) or (II), or polymers comprising such compounds assubstituents. The kits further contain anti-IgM antibodies (or anti-IgMantibody fragments) carrying a suitable label, or anti-IgM antibodiesand second antibodies carrying such a suitable label, and reagents orequipment to detect the label, e.g. reagents reacting with enzymes usedas labels and indicating the presence of such a label by a colourformation or fluorescence, standard equipment, such as microtiterplates, pipettes and the like, standard solutions and wash solutions.

The ELISA can be also designed in a way that patient blood or serumsamples are used for the coating of microtiter plates with thesubsequent detection of anti-MAG antibodies with labelled compounds offormula (I) or (II), or labelled polymers comprising such compounds assubstituents. The label is either directly detectable or indirectlydetectable via an antibody.

The polymer carrying compounds of formula (I) or (II) of the inventionbinds to the pathogenic anti-MAG IgM antibodies and potentiallydownregulates the anti-MAG IgM antibody production. It allows anantigen-specific treatment for anti-MAG neuropathy patients.

Furthermore the invention relates to a pharmaceutical compositioncomprising a compound of formula (I) or (II), or a polymer carryingcompounds of formula (I) or (II) of the invention.

Pharmaceutical compositions for parenteral administration, such assubcutaneous, intravenous, intrahepatic or intramuscular administration,to warm-blooded animals, especially humans, are considered. Thecompositions comprise the active ingredient(s) alone or, preferably,together with a pharmaceutically acceptable carrier. The dosage of theactive ingredient(s) depends upon the age, weight, and individualcondition of the patient, the individual pharmacokinetic data, and themode of administration.

For parenteral administration preference is given to the use ofsuspensions or dispersions of the carbohydrate polymer of the invention,especially isotonic aqueous dispersions or suspensions which, forexample, can be made up shortly before use. The pharmaceuticalcompositions may be sterilized and/or may comprise excipients, forexample preservatives, stabilizers, wetting agents and/or emulsifiers,solubilizers, viscosity-increasing agents, salts for regulating osmoticpressure and/or buffers and are prepared in a manner known per se, forexample by means of conventional dissolving and lyophilizing processes.

Suitable carriers for enteral administration, such as nasal, buccal,rectal or oral administration, are especially fillers, such as sugars,for example lactose, saccharose, mannitol or sorbitol, cellulosepreparations, and/or calcium phosphates, for example tricalciumphosphate or calcium hydrogen phosphate, and also binders, such asstarches, for example corn, wheat, rice or potato starch,methylcellulose, hydroxypropyl methylcellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired,disintegrators, such as the above-mentioned starches, also carboxymethylstarch, crosslinked polyvinylpyrrolidone, alginic acid or a saltthereof, such as sodium alginate. Additional excipients are especiallyflow conditioners and lubricants, for example silicic acid, talc,stearic acid or salts thereof, such as magnesium or calcium stearate,and/or polyethylene glycol, or derivatives thereof.

Tablet cores can be provided with suitable, optionally enteric, coatingsthrough the use of, inter alia, concentrated sugar solutions which maycomprise gum arabic, talc, polyvinyl-pyrrolidone, polyethylene glycoland/or titanium dioxide, or coating solutions in suitable organicsolvents or solvent mixtures, or, for the preparation of entericcoatings, solutions of suitable cellulose preparations, such asacetylcellulose phthalate or hydroxypropyl-methylcellulose phthalate.Dyes or pigments may be added to the tablets or tablet coatings, forexample for identification purposes or to indicate different doses ofactive ingredient(s).

Pharmaceutical compositions for oral administration also include hardcapsules consisting of gelatin, and also soft, sealed capsulesconsisting of gelatin and a plasticizer, such as glycerol or sorbitol.The hard capsules may contain the active ingredient in the form ofgranules, for example in admixture with fillers, such as corn starch,binders, and/or glidants, such as talc or magnesium stearate, andoptionally stabilizers. In soft capsules, the active ingredient ispreferably dissolved or suspended in suitable liquid excipients, such asfatty oils, paraffin oil or liquid polyethylene glycols or fatty acidesters of ethylene or propylene glycol, to which stabilizers anddetergents, for example of the polyoxy-ethylene sorbitan fatty acidester type, may also be added.

Pharmaceutical compositions suitable for rectal administration are, forexample, suppositories that consist of a combination of the activeingredient and a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols.

The mentioned pharmaceutical compositions according to the invention maycontain separate tablets, granules or other forms of orally acceptableformulation of the active ingredients, or may contain a mixture ofactive ingredients in one suitable pharmaceutical dosage form, asdescribed above. In particular the separate orally acceptableformulations or the mixture in one suitable pharmaceutical dosage formmay be slow release and controlled release pharmaceutical compositions.

The pharmaceutical compositions comprise from approximately 1% toapproximately 95% active ingredient or mixture of active ingredients,single-dose administration forms comprising in the preferred embodimentfrom approximately 20% to approximately 90% active ingredient(s) andforms that are not of single-dose type comprising in the preferredembodiment from approximately 5% to approximately 20% activeingredient(s).

The invention also relates to the mentioned pharmaceutical compositionsas medicaments in the treatment of anti-MAG neuropathy.

The present invention relates furthermore to a method of treatment ofanti-MAG neuropathy, which comprises administering a compositionaccording to the invention in a quantity effective against said disease,to a warm-blooded animal requiring such treatment. The pharmaceuticalcompositions can be administered prophylactically or therapeutically,preferably in an amount effective against the said diseases, to awarm-blooded animal, for example a human, requiring such treatment. Inthe case of an individual having a bodyweight of about 70 kg the dailydose administered is from approximately 0.01 g to approximately 5 g,preferably from approximately 0.25 g to approximately 1.5 g, of theactive ingredients in a composition of the present invention.

The following Examples serve to illustrate the invention withoutlimiting the invention in its scope.

EXAMPLES

General Methods

NMR spectra were obtained on a Bruker Avance DMX-500 (500 MHz)spectrometer.

Assignment of ¹H and ¹³C NMR spectra was achieved using 2D methods (COSYand HSQC). Chemical shifts are expressed in ppm using residual CHCl₃,CHD₂OD or HDO as references. Optical rotations were measured on aPerkin-Elmer polarimeter 341. IR spectra were recorded using aPerkin-Elmer Spectrum One FT-IR spectrometer. Electron spray ionizationmass spectra (ESI-MS) were obtained on a Waters micromass ZQ. HRMSanalysis was carried using an Agilent 1100 LC equipped with a photodiodearray detector and a Micromass QTOF I equipped with a 4 GHz digital-timeconverter. Reactions were monitored by TLC using glass plates coatedwith silica gel 60 F₂₅₄ (Merck) and visualized by using UV light and/orby charring with mostain (a 0.02 M solution of ammonium cerium sulfatedihydrate and ammonium molybdate tetrahydrate in aq 10% H₂SO₄). Columnchromatography was performed on silica gel (Fluka C60 40/60) or RP-18(Merck LiChroprep® RP-18 40/60). Methanol (MeOH) was dried by refluxingwith sodium methoxide and distillation. Pyridine was dried overactivated molecular sieves (4 Å). Dimethylformamide (DMF) was purchasedfrom Acros (99.8%, extra dry, over molecular sieves). Dichloromethane(DCM), toluene and hexane were dried by filtration over Al₂O₃ (Fluka,type 5016A basic). Molecular sieves (4 Å) were activated in vacuo at500° C. for 1 h immediately before use. Centrifugations were carried outwith an Eppendorf Centrifuge 5804 R. rt=room temperature.

The three compounds for the biological evaluation (1, 2 and 25) weresynthesized according to Scheme 1 and 2. All reagents were bought fromSigma Aldrich or Acros. The GlcA-Gal disaccharides 5 were obtained byreacting the activated GlcA donor 3 (C. Coutant and J.-C. Jacquinet, JChem Soc Perkin Trans I, 1995, 1573-1581) and the selectively protectedGal acceptor 4 (F. Belot et al., Synlett 2003, 1315-1318) withtrimethylsilyl trifluoromethanesulfonate (TMSOTf) as promoter.Deprotection of the ester groups with LiOH in tetrahydrofuran(THF)/water (H₂O) yielded 6. Disaccharides 2 were obtained by catalytichydrogenation. The 3′-unprotected disaccharides 7 were synthesized via alactonization/methanolysis procedure published by A. V. Kornilov(Carbohydrate Research 2000, 329:717-730). Subsequent sulfation with thesulfate-pyridine complex (SO₃·Py) in N,N-dimethylformamide (DMF) gave3-O-sulfated disaccharide 8 (65%). Final deprotection by catalytichydrogenation followed by hydrolysis and treatment with Na⁺cation-exchange resin afforded the desired sulfated disaccharides 1.

For the synthesis of the carbohydrate polymer 25, the sulfated monomer21 was prepared (Scheme 1). It contains a 4-(2-aminoethyl)phenylaglycone instead of para-methoxyphenyl present in 1. The additionalprimary amino group was required for the coupling to the polylysinepolymer. For its synthesis, 4-(2-azidoethyl)phenol (9) wasgalactosylated with the trichloroacetimidate donor 10 (R. Burkowski etal., Eur J Org Chem 2001, 2697-2705). Acceptor 9 was obtained byamine-azide interconversion (A. Titz et al., Tet. Letters 2006,47:2383-2385) from tyrosine. Deacetylation under Zempén conditions(giving 12), followed by the formation of the 3,4-isopropylidenederivative 13, dibenzylation (results in 14), acid-catalyzed cleavage ofthe acetonide (gives 15) and mono-benzoylation yielded galactoside 16.For the remaining steps to the monosulfated disaccharide 21 a similarreaction sequence as already applied for the synthesis of disaccharide 1was applied, except for the benzylation which was carried out underphase transfer catalysis using 50% aqueous NaOH/DCM and 18-crown-6ether. The free amino group in 21 was then reacted withthiobutyrolactone and triethylamine (TEA) in DMF to give compound 22 in59% yield, ready for coupling to the polylysine polymer.

For this purpose, the commercial polylysine polymer 23 was acylated,giving 24 in 96% yield (G. Thoma et al., J Am Chem Soc 1999,121:5919-5929) before it was coupled to a substochiometric amount of theminimal HNK-1 epitope 22 (0.4 eq). To improve the water solubility ofthe glycosylated polylysine polymer, the remaining chloroacetamidegroups were capped with an excess of thioglycerol. Purification byultrafiltration (Sartorius Stedim Vivaspin 6, molecular weight cutoff,5000) yielded glycopolymer 25 in 70% yield.

4-Methoxyphenyl (Methyl2,3,4-Tri-O-Benzoyl-β-D-Glucopyranuronate)-(1→3)-4-O-Benzoyl-2,6-Di-O-Benzyl-β-D-Galactopyranoside(5).

Under argon 3 (1.12 g, 1.68 mmol), 4a (800 mg, 1.40 mmol) and activated4 Å molecular sieves (1.2 g) were suspended in DCM (30 mL). The mixturewas stirred for 1 h at rt and then cooled to 0 ° C. TMSOTf (38.1 μL,0.21 mmol) was added dropwise. The reaction mixture was allowed to warmto rt overnight, and was then neutralized with TEA (100 μL) andconcentrated. The residue was purified by chromatography (petroleumether/EtOAc, 9:1 to 7:3) to yield 5 (1.21 g, 1.13 mmol, 81%) as a whitesolid.

[α]_(D) ²⁰+28.4 (c 1.01, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 3.59 (dd,J=7.2, 10.1 Hz, 1H, H-6a), 3.65 (s, 3H, OMe), 3.69 (dd, J=4.8, 10.1 Hz,1H, H-6b), 3.74 (s, 3H, OMe), 3.93 (dd, J=7.8, 9.5 Hz, 1H, H-2), 3.96(dd, J=5.1, 6.9 Hz, 1H, H-5), 4.12 (d, J=9.8 Hz, 1H, H-5′), 4.20 (dd,J=3.5, 9.6 Hz, 1H, H-3), 4.46 (A, B of AB, J=11.5 Hz, 2H, CH₂Ph), 4.51,4.90 (A, B of AB, J=10.5 Hz, 2H, CH₂Ph), 4.94 (d, J=7.8 Hz, 1H, H-1),5.36 (d, J=7.5 Hz, 1H, H-1′), 5.44 (dd, J=7.5, 9.2 Hz, 1H, H-2′), 5.66(t, J=9.6 Hz, 1H, H-4), 5.72-5.79 (m, 2H, H-3′, H-4′), 6.76, 7.00 (AA′,BB′ of AA′BB′, J=9.1 Hz, 4H, C₆H₄), 7.19-7.44, 7.47-7.51, 7.54-7.61,7.75-7.78, 7.87-7.91, 8.03-8.08 (m, 30H, 6 C₆H₅); ¹³C NMR (126 MHz,CDCl₃): δ 52.88, 55.64 (2 OMe), 69.07 (C-6), 70.01 (C-4), 70.05 (C-4′),71.76 (C-2′), 72.17 (C-3′), 72.90 (C-5′), 73.54 (C-5), 73.72, 75.23 (2CH₂Ph), 76.16 (C-3), 79.86 (C-2), 100.29 (C-1′), 102.73 (C-1), 114.57,118.19 (4C, C₆H₄), 127.69, 127.78, 128.00, 128.09, 128.30, 128.37,128.43, 128.59, 128.71, 128.89, 129.05, 129.58, 129.77, 129.82, 129.92,130.11, 132.91, 133.08, 133.27, 133.39, 137.88, 137.90 (36C, 6 C₆H₅),151.33, 155.33 (2C, C₆H₄), 164.45, 165.00, 165.52, 165.63, 167.15 (5CO); ESI-MS: m/z: calcd for C₆₂H₅₆NaO₁₇ [M+Na]⁺: 1095.35, found:1095.48.

4-Methoxyphenyl(β-D-Glucopyranuronate)-(1→3)-2,6-Di-O-Benzyl-β-D-Galactopyranoside

Compound 5 (810 mg, 0.76 mmol) was suspended in THF (7 mL) and thesuspension was cooled to −10° C. Then 2 M aq LiOH (5 mL) was addeddropwise. The reaction mixture was stirred overnight and allowed to warmto rt. The solvents were evaporated, the residue was taken up in THF/H₂O(2:3, 8 mL) and treated with TFA (4 mL) for 30 min. The mixture wasevaporated to dryness and the residue was purified by reversed-phasechromatography (RP-18, MeOH/water, 0:1 to 2:1) to give 6 (0.47 g, 0.73mmol, 97%) as a white solid.

[α]_(D) ²⁰ −43.2 (c 1.00, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 3.30-3.41(m, 2H, H-2′, H-3′), 3.49 (t, J=8.9 Hz, 1 H, H-4′), 3.66 (s, 3H, OMe),3.68 (d, J=5.9 Hz, 2H, H-6a, H-6b), 3.72 (d, J=9.7 Hz, 1 H, H-5′), 3.76(d, J=5.9 Hz, 1 H, H-5), 3.79 (dd, J=3.3, 9.9 Hz, 1 H, H-3), 3.87 (m,1H, H-2), 4.00 (d, J=2.7 Hz, 1H, H-4), 4.48 (s, 2H, CH₂Ph), 4.70 (d,J=7.4 Hz, 1 H, H-1′), 4.84 (d, J=7.7 Hz, 1H, H-1), 4.87 (s, 2H, CH₂Ph),6.73, 6.97 (AA′, BB′ of AA′BB′, J=9.0 Hz, 4H, C₆H₄), 7.17-7.28 (m, 8H, 2C₆H₅), 7.38 (d, J=7.1 Hz, 2H, 2 C₆H₅); ¹³C NMR (126 MHz, CD₃OD): δ 56.10(OMe), 70.37 (C-4), 70.72 (C-6), 73.35 (C-4′), 74.37 (CH₂Ph), 74.85(C-2′), 75.00 (C-5), 76.22 (C-5′), 76.46 (CH₂Ph), 77.35 (C-3′), 80.11(C-2), 82.20 (C-3), 103.87 (C-1), 105.59 (C-1′), 115.58, 119.23 (4C,C₆H₄), 128.66, 128.76, 128.79, 129.31, 129.41, 129.77, 139.76, 139.96(12C, 2 C₆H₅), 153.05, 156.67 (2C, C₆H₄), 173.01 (CO); ESI-MS: m/z:calcd for C₃₃H₃₈NaO₁₃[M+Na]⁺: 665.23, found: 665.23.

4-Methoxyphenyl (Sodiumβ-D-Glucopyranuronate)-(1→3)-β-D-Galactopyranoside (2)

Compound 6 (205 mg, 0.31 mmol) and Pd(OH)₂/C (42 mg, 20%) were suspendedin MeOH/H₂O (10:1, 5 mL) under argon. The mixture was stirred overnightunder an atmosphere of hydrogen (1 atm), then the catalyst was filteredoff through a pad of Celite. The Celite was washed with a MeOH/H₂Ogradient (6×10 mL, 10:0, 8:2, 6:4, 4:6, 2:8, 0:10). The filtrate wasconcentrated and passed over a Dowex® 50X8 (Na⁺) ion-exchange column.After concentration the residue was purified by reversed-phasechromatography (RP-18, water) followed by P2 size-exclusionchromatography to give 2 (148 mg, 0.31 mmol, 96%) as a white solid.

[α]_(D) ²⁰ −40.7 (c 1.00, H₂O); ¹H NMR (500 MHz, D₂O): δ 3.43 (t, J=8.3Hz, 1H, H-2′), 3.48-3.56 (m, 2H, H-3′, H-5′), 3.67-3.81 (m, 7H, H-5,H-6, H-4′, OMe), 3.83 (dd, J=2.9, 9.8 Hz, 1 H, H-3), 3.90 (dd, J=8.0 Hz,1 H, H-2), 4.22 (d, J=2.5 Hz, 1H, H-4), 4.68 (d, J=7.7 Hz, 1H, H-1′),4.95 (d, J=7.9 Hz, 1H, H-1), 6.94, 7.09 (AA′, BB′ of AA′BB′, J=9.0 Hz,4H, C₆H₄); ¹³C NMR (126 MHz, D₂O): δ 55.71 (OMe), 60.70 (C-6), 67.94(C-4), 69.63 (C-2), 71.73 (C-3′), 73.09 (C-2′), 75.05 (C-5′), 75.25(C-5), 76.18 (C-4′), 82.37 (C-3), 101.29 (C-1), 103.61 (C-1′), 114.96,118.10, 150.84, 154.61 (6C, C₆H₄), 175.92 (CO); HRMS: m/z: calcd forC₁₉H₂₆NaO₁₃[M+H]⁺: 485.1271, found: 485.1276.

4-Methoxyphenyl (Methyl2,4-Di-O-Acetyl-β-D-Glucopyranuronate)-(1→3)-4-O-Acetyl-2,6-Di-O-Benzyl-β-D-Galactopyranoside(7)

A solution of 6 (470 mg, 0.73 mmol) in Ac₂O (10 mL) was stirred at 80°C. for 90 min and then cooled to rt. Pyridine (6 mL) and DMAP (15 mg)were added and the reaction mixture was stirred for 3 days. The solventswere co-evaporated with toluene (5×5 mL). The residue dissolved in DCM(50 mL) and extracted with brine (50 mL) and water (50 mL). The organicphase was dried over Na₂SO₄ and filtered through cotton wool. Afterevaporation of the solvent the residue was dissolved in dry MeOH (14 mL)and anhydrous NaOAc (90 mg) was added. The mixture was stirredovernight, neutralized with Amberlyste® 15 (H⁺) ion-exchange resin andfiltered. The filtrate was concentrated and the residue purified byflash chromatography (petroleum ether/EtOAc, 2:1 to 1:1) to yield 7 (334mg, 0.43 mmol, 57%) as a yellowish solid.

[α]_(D) ²⁰+34.3 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.92, 2.01,2.04 (3s, 9H, 3 OAc), 3.48 (dd, J=7.1, 10.1 Hz, 1H, H-6a), 3.55 (dd,J=4.8, 10.1 Hz, 1H, H-6b), 3.60 (m, 1 H, H-3′), 3.66, 3.69 (2s, 6H, 2OMe), 3.77 (dd, J=5.4, 7.0 Hz, 1H, H-5), 3.80 (d, J=9.8 Hz, 1H, H-5′),3.83 (dd, J=7.6, 9.7 Hz, 1H, H-2), 3.89 (dd, J=3.5, 9.7 Hz, 1H, H-3),4.43 (A, B of AB, J=11.6 Hz, 2H, CH₂Ph), 4.64 (A of AB, J=11.5 Hz, 1H,CH₂Ph), 4.81 (d, J=7.6 Hz, 1H, H-1), 4.83-4.87 (m, H-1′, H-2′), 4.97 (Bof AB, J=11.5 Hz, 1H, CH₂Ph), 5.06 (t, J=9.5 Hz, 1H, H-4′), 5.36 (d,J=3.2 Hz, 1H, H-4), 6.72, 6.96 (AA′, BB′ of AA′BB′, J=9.1 Hz, 4H, C₆H₄),7.18-7.31 (m, 10H, 2 C₆H₅); ¹³C NMR (126 MHz, CDCl₃): δ 20.72, 20.76,20.80 (3 COCH₃), 52.81, 55.63 (2 OMe), 68.95 (C-6), 69.33 (C-4), 71.87(C-4′), 72.54 (C-5), 73.04 (C-5′), 73.26 (C-3′), 73.70 (CH₂Ph), 73.79(C-2′), 75.31 (CH₂Ph), 77.24 (C-3), 79.26 (C-2), 100.15 (C-1′), 102.65(C-1), 114.56, 118.24 (4C, C₆H₄), 127.76, 127.83, 127.98, 128.04,128.41, 128.53, 137.87, 138.00 (12C, 2 C₆H₅), 151.35, 155.35 (2C, C₆H₄),167.46, 170.15, 170.36, 170.38 (4 CO); ESI-MS: m/z: calcd forC₄₀H₄₆NaO₁₆ [M+Na]⁺: 805.28, found: 805.34.

4-Methoxyphenyl (Methyl2,4-Di-O-Acetyl-3-O-Sulfo-β-D-Glucopyranuronate)-(1→3)-4-O-Acetyl-2,6-Di-O-Benzyl-β-D-Galactopyranoside,Sodium Salt (8)

Compound 7 (334 mg, 0.43 mmol) was dissolved in DMF (5 mL) and SO₃·Py(370 mg, 2.34 mmol) was added. The mixture was stirred for 2 h at rt,then the reaction was quenched by stirring with NaHCO₃ (320 mg, 3.77mmol) for 2 h. The solid was filtered off and the filter was washed withMeOH. The filtrate was passed over a Dowex® 50×8 (Na⁺) ion-exchangecolumn, concentrated and the residue was purified by flashchromatography (DCM/MeOH, 1:0 to 9:1) to give 8 (237 mg, 0.28 mmol, 65%)as a yellowish solid. During concentration after the flashchromatography a few drops of 0.1 M aq NaOH were added.

[α]_(D) ²⁰ −10.4 (c 1.01, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 1.89, 2.03,2.06 (3s, 9H, 3 OAc), 3.48 (dd, J=7.4, 10.4 Hz, 1H, H-6a), 3.59 (dd,J=4.4, 10.5 Hz, 1H, H-6b), 3.69, 3.72 (2s, 6H, 2 OMe), 3.77 (dd, J=7.8,9.6 Hz, 1H, H-2), 3.98 (dd, J=4.5, 7.4 Hz, 1H, H-5), 4.03 (m, 1H, H-3),4.05 (d, J=10.2 Hz, 1H, H-5′), 4.46, 4.49 (A, B of AB, J=11.6 Hz, 2H,CH₂Ph), 4.60 (t, J=9.3 Hz, 1H, H-3′), 4.73, 4.92 (A, B of AB, J=11.8 Hz,2H, CH₂Ph), 4.94 (d, J=7.5 Hz, 1H, H-2′), 4.96 (d, J=7.9 Hz, 1H, H-1′),4.99 (d, J=8.0 Hz, 1 H, H-1), 5.06 (m, 1H, H-4′), 5.40 (d, J=3.6 Hz, 1H,H-4), 6.77, 7.00 (AA′, BB′ of AA′BB′, J=9.1 Hz, 4H, C₆H₄), 7.23-7.35 (m,8H, 2 C₆H₅), 7.39 (d, J=7.2 Hz, 2H, 2 C₆H₅); ¹³C NMR (126 MHz, CD₃OD): δ19.32, 19.23, 19.64 (3 COCH₃), 51.68, 54.52 (2 OMe), 68.73 (C-6), 69.50(C-4), 69.80 (C-4′), 71.36 (C-2′), 71.91 (C-5′), 72.52 (C-5), 72.83,74.69 (2 CH₂Ph), 77.50 (C-3′), 78.57 (C-3), 78.59 (C-2), 100.04 (C-1),102.01 (C-1′), 114.03, 117.64 (4C, C₆H₄), 127.14, 127.23, 127.36,127.57, 127.81, 127.89, 138.02, 138.23 (12C, 2 C₆H₅), 151.29, 155.26(2C, C₆H₄), 167.77, 170.07, 170.17, 170.64 (4 CO); ESI-MS: m/z: calcdfor C₄₀H₄₆O₁₉S [M]⁺: 862.24, found: 862.42.

4-Methoxyphenyl(Disodium-3-O-Sulfo-β-D-Glucopyranuronate)-(1→3)-β-D-Galacto-Pyranoside(1)

Compound 8 (237 mg, 0.28 mmol) and Pd(OH)₂/C (48 mg, 20%) were suspendedin MeOH/H₂O (10:1, 5 mL) under argon. The reaction mixture was stirredfor 9 h under an atmosphere of hydrogen (1 atm). The catalyst wasfiltered off through a pad of Celite and the pad was washed with aMeOH/H₂O gradient (6×10 mL, 10:0, 8:2, 6:4, 4:6, 2:8, 0:10). Thefiltrate was concentrated and the residue was dissolved in MeOH/H₂O(1:1, 8 mL). Then 1 M aq LiOH (6.5 mL) was added at −10° C. and thereaction mixture was allowed to warm to rt over 3 h, neutralized withAmberlyste® 15 (H⁺) ion-exchange resin, filtered and concentrated. Theresidue was purified by reversed-phase chromatography (RP-18, water) andpassed over a Dowex® 50X8 (Na⁺) ion-exchange column. Final purificationby P2 size-exclusion chromatography yielded 1 (142 mg, 0.24 mmol, 88%)as a solid.

[α]D²⁰ −19.2 (c 1.00, H₂O); ¹H NMR (500 MHz, D₂O): δ 3.63 (dd, J=8.0,9.2 Hz, 1H, H-2′), 3.73 (m, 1H, H-4′), 3.75-3.81 (m, 6H, H-5, H-6, OMe),3.85 (d, J=10.0 Hz, 1H, H-5′), 3.89 (dd, J=3.2, 9.9 Hz, 1H, H-3), 3.94(dd, J=7.7, 9.8 Hz, 1H, H-2), 4.24 (d, J=3.1 Hz, 1H, H-4), 4.40 (t,J=9.2 Hz, 1H, H-3′), 4.81 (d, J=7.9 Hz, 1H, H-1′), 4.97 (d, J=7.7 Hz,1H, H-1), 6.96, 7.11 (AA′, BB′ of AA′BB′, J=9.2 Hz, 4H, C₆H₄); ¹³C NMR(126 MHz, D₂O): δ 55.82 (OMe), 60.62 (C-6), 67.95 (C-4), 69.55 (C-2),70.42 (C-4′), 71.86 (C-2′), 74.92 (C-5), 75.82 (C-5′), 82.49 (C-3),83.30 (C-3′), 101.43 (C-1), 103.17 (C-1′), 115.02, 118.14, 150.89,154.59 (6C, C₆H₄), 175.48 (CO); HRMS: m/z: calcd for C₁₉H₂₅Na₂O₁₆S[M+H]⁺: 587.0659, found: 587.0665.

4-(2-Azidoethyl)Phenol (9)

Tyramine (3.43 g, 25.0 mmol), NaHCO₃ (7.80 g, 92.8 mmol) and CuSO₄·5H₂O(0.22 g, 0.9 mmol) were dissolved in water (30 mL). Triflic azide stocksolution (40 mL), which was prepared according to Titz A. et al.,Tetrahedron Letters 47:2383-2385 (2006), and MeOH (190 mL) were added togive a homogeneous mixture. The mixture was stirred at rt overnight,then diluted with water (150 mL) and extracted with EtOAc (3×150 mL).The organic layer was dried over Na₂SO₄ and the solvents wereevaporated. The residue was purified by flash chromatography (petroleumether/EtOAc, 1:0 to 4:1) to yield 9 (quant.) as colorless oil.

¹H NMR (500 MHz, CDCl₃): δ 2.81 (t, J=7.3 Hz, 2H, CH₂CH₂N₃), 3.44 (t,J=7.2 Hz, 2H, CH₂CH₂N₃), 6.77, 7.07 (AA′, BB′ of AA′BB′, J=8.5 Hz, 4H,C₆H₄); ¹³C NMR (126 MHz, CDCl₃): δ 34.50 (CH₂CH₂N₃), 52.72 (CH₂CH₂N₃),115.53, 129.96, 130.22, 154.39 (6C, C₆H₄); IR (film): 2105 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl 2,3,4,6-Tetra-O-Acetyl-β-D-Galactopyranoside (11)

To an ice-cooled suspension of 10 (8.30 g, 17.5 mmol) (Bukowski R etal., European Journal of Organic Chemistry 2001:2697-2705) and 4 Amolecular sieves (3 g) in DCM (40 mL) was added 9 (4.00 g, 24.5 mmol) inDCM (40 mL) under argon. TfOH (0.45 mL, 2.5 mmol) was added dropwise andthe reaction mixture was allowed to warm to rt overnight. Afterquenching with TEA (0.8 mL) the suspension was filtered and the filtratewas concentrated. The residue was purified by flash chromatography(petroleum ether/EtOAc, 9:1 to 3:2) to yield 11 (4.58 g, 9.28 mmol, 53%)as oil.

[α]_(D) ²⁰+6.1 (c 1.10, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.98, 2.02,2.06, 2.15 (4s, 12H, 4 OAc), 2.82 (t, J=7.2 Hz, 2H, CH₂CH₂N₃), 3.45 (t,J=7.1 Hz, 2H, CH₂CH₂N₃), 4.02 (t, J=6.6 Hz, 1H, H-5), 4.13 (dd, J=6.3,11.3 Hz, 1H, H-6a), 4.20 (dd, J=6.9, 11.2 Hz, 1H, H-6b), 4.99 (d, J=8.0Hz, 1H, H-1), 5.08 (dd, J=3.4, 10.5 Hz, 1H, H-3), 5.40-5.48 (m, 2H, H-2,H-4), 6.93, 7.12 (AA′, BB′ of AA′BB′, J=8.6 Hz, 4H, C₆H₄); ¹³C NMR (126MHz, CDCl₃): δ 20.58, 20.65, 20.65, 20.73 (4 COCH₃), 34.52 (CH₂CH₂N₃),52.51 (CH₂CH₂N₃), 61.36 (C-6), 66.89 (C-4), 68.67 (C-2), 70.85 (C-3),71.01 (C-5), 99.78 (C-1), 117.19, 129.87, 133.01, 155.85 (6C, C₆H₄),169.40, 170.13, 170.26, 170.36 (4 CO); ESI-MS: m/z: calcd forC₂₂H₂₇N₃NaO₁₀ [M+Na]⁺: 516.17, found: 516.19; IR (film): 2101 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl β-D-Galactopyranoside (12)

A solution of 11 (4.58 g, 9.28 mmol) in MeOH (45 mL) was treated with 1M NaOMe/MeOH (4.5 mL) under argon overnight. After neutralization withAmberlite® IR-120 (H⁺) ion-exchange resin, the solvent was evaporatedand the residue was purified by flash chromatography (DCM/MeOH, 1:0 to4:1) to give 12 (2.86g, 8.79 mmol, 95%) as an oil.

[α]_(D) ²⁰ −38.1 (c 1.00, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 2.85 (t,J=7.1 Hz, 2H, CH₂CH₂N₃), 3.49 (t, J=7.1 Hz, 2H, CH₂CH₂N₃), 3.60 (dd,J=3.4, 9.7 Hz, 1H, H-3), 3.70 (m, 1H, H-5), 3.75-3.85 (m, 3H, H-2, H-6),3.93 (d, J=3.2 Hz, 1H, H-4), 4.86 (d, J=7.8 Hz, 1H, H-1), 7.09, 7.20(AA′, BB′ of AA′BB′, J=8.6 Hz, 4H, C₆H₄); ¹³C NMR (126 MHz, CD₃OD): δ35.49 (CH₂CH₂N₃), 53.75 (CH₂CH₂N₃), 62.44 (C-6), 70.25 (C-4), 72.34(C-2), 74.89 (C-3), 76.96 (C-5), 103.11 (C-1), 118.00, 130.82, 133.65,158.08 (6C, C₆H₄); ESI-MS: m/z: calcd for C₁₄H₁₉N₃NaO₆[M+Na]⁺: 348.13,found: 348.04; IR (film): 2112 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl 3,4-Isopropylidene-β-D-Galactopyranoside (13)

To a solution of 12 (2.86 g, 8.79 mmol) in DMF (30 mL) were added2,2-dimethoxy-propane (2.50 mL, 19.3 mmol) and p-TsOH (37 mg) underargon. After stirring overnight at 80° C., the reaction mixture wasneutralized with TEA (0.5 mL) and the solvents were evaporated. Theresidue was purified by flash chromatography (petroleum ether+0.5%TEA/EtOAc, 1:2 to 0:1) to yield 13 (2.39 g, 6.55 mmol, 75%) as an oil.

[α]_(D) ²⁰ −22.4 (c 1.10, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.34, 1.53(2s, 6H, Me₂C), 2.42 (s, 2H, 2 OH), 2.81 (t, J=7.1 Hz, 2H, CH₂CH₂N₃),3.44 (t, J=7.2 Hz, 2H, CH₂CH₂N₃), 3.78-3.85 (m, 2H, H-2, H-6a),3.93-4.00 (m, 2H, H-6b, H-5), 4.14-4.21 (m, 2H, H-3, H-4), 4.78 (d,J=8.2 Hz, 1H, H-1), 6.95, 7.12 (AA′, BB′ of AA′BB′, J=8.5 Hz, 4H, C₆H₄);¹³C NMR (126 MHz, CDCl₃): δ 26.33, 28.10 (C(CH₃)₂), 34.54 (CH₂CH₂N₃),52.53 (CH₂CH₂N₃), 62.29 (C-6), 73.31 (C-2), 73.69 (C-5), 73.87 (C-4),78.89 (C-3), 100.33 (C-1), 110.69 (C(CH₃)₂), 116.89, 129.95, 132.63,155.78 (6C, C₆H₄); ESI-MS: m/z: calcd for C₁₇H₂₃N₃NaO₆[M+Na]⁺: 388.16,found: 388.06; IR (film): 2099 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl2,6-Di-O-Benzyl-3,4-Isopropylidene-β-D-Galactopyranoside (14)

Compound 13 (1.02 g, 2.78 mmol) was dissolved in DCM (15 mL). 15-Crown-5(55 μL, 0.28 mmol), 50% aq NaOH (37.5 mL) and benzylbromide (3.30 mL,27.8 mmol) were added and the biphasic mixture was stirred overnightunder reflux at 60° C. The reaction mixture was neutralized with 4 M aqHCI. The organic layer was separated and the aqueous phase extractedwith DCM (2×50 mL) and. The combined organic layers were concentratedand the residue was purified by flash chromatography (petroleumether+0.5% TEA/EtOAc, 1:0 to 3:1) to give 14 (1.26 g, 2.31 mmol, 83%) asa white solid.

[α]_(D) ²⁰ +8.4 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.28, 1.34(2s, 6H, Me₂C), 2.76 (t, J=7.3 Hz, 2H, CH₂CH₂N₃), 3.37 (t, J=7.3 Hz, 2H,CH₂CH₂N₃), 3.60 (dd, J=6.8, 7.9 Hz, 1H, H-2), 3.69-3.80 (m, 2H, H-6),3.97 (ddd, J=1.8, 4.7, 6.8 Hz, 1H, H-5), 4.13 (dd, J=2.0, 5.7 Hz, 1H,H-4), 4.18 (m, 1H, H-3), 4.46, 4.54 (A, B of AB, J=11.8 Hz, 2H, CH₂Ph),4.78-4.85 (m, 3H, CH₂Ph, H-1), 6.69, 7.03 (AA′, BB′ of AA′BB′, J=8.6 Hz,4H, C₆H₄), 7.15-7.28 (m, 8H, 2 C₆H₅), 7.34 (d, J=7.4 Hz, 2H, 2 C₆H₅);¹³C NMR (126 MHz, CDCl₃): δ 26.41, 27.81 (C(CH₃)₂), 34.62 (CH₂CH₂N₃),52.62 (CH₂CH₂N₃), 69.60 (C-6), 72.72 (C-5), 73.67 (C-4), 73.69 (2C, 2CH₂Ph), 79.08 (C-3), 79.26 (C-2), 101.09 (C-1), 110.27 (C(CH₃)₂), 117.23(2C, C₆H₄), 127.63, 127.69, 127.72, 128.26, 128.32, 128.40 (8C, 2 C₆H₅),129.80 (2C, C₆H₄), 132.19, 138.14 (2 C₆H₅), 138.29, 156.26 (C₆H₄);ESI-MS: m/z: calcd for C₃₁H₃₅N₃NaO₆[M+Na]⁺: 568.25, found: 568.21; IR(KBr): 2096 cm⁻¹ (N₃).δ

4-(2-Azidoethyl)Phenyl 2,6-Di-O-Benzyl-β-D-Galactopyranoside (15)

A solution of 14 (1.26 g, 2.31 mmol) in 90% aq AcOH (50 mL) was stirredat 60° C. stirred overnight. The solvents were evaporated and theresidue was purified by flash chromatography (DCM/MeOH, 1:0 to 9:1) togive 15 (1.17 g, 2.31 mmol, quant) as an oil.

[α]_(D) ²⁰ −9.9 (c 1.10, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 2.76 (t,J=7.3 Hz, 2H, CH₂CH₂N₃), 3.38 (t, J=7.3 Hz, 2H, CH₂CH₂N₃), 3.59 (dd,J=3.3, 9.5 Hz, 1H, H-3), 3.62-3.76 (m, 4H, H-2, H-5, H6), 3.92 (d, J=3.2Hz, 1H, H-4), 4.48 (s, 2H, CH₂Ph), 4.69 (A of AB, J=11.5 Hz, 1H, CH₂Ph),4.87 (d, J=7.7 Hz, 1H, H-1), 4.96 (B of AB, J=11.5 Hz, 1H, CH₂Ph), 6.97,7.05 (AA′, BB′ of AA′BB′, J=8.5 Hz, 4H, C₆H₄), 7.15-7.31 (m, 10H, 2C₆H₅); ¹³C NMR (126 MHz, CDCl₃): δ 34.58 (CH₂CH₂N₃), 52.59 (CH₂CH₂N₃),68.92 (C-4), 69.41 (C-6), 73.20 (C-3), 73.74 (C-5), 73.81, 74.91 (2CH₂Ph), 78.87 (C-2), 101.86 (C-1), 117.19 (2C, C₆H₄), 127.75, 127.83,128.03, 128.27, 128.47, 128.60 (8C, 2 C₆H₅), 129.83 (2C, C₆H₄), 132.33,137.87 (2 C₆H₅), 138.14, 156.13 (C₆H₄); ESI-MS: m/z: calcd forC28H₃₁N₃NaO₆[M+Na]⁺: 528.22, found: 528.22; IR (film): 2098 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl 4-O-benzoyl-2,6-di-O-benzyl-β-D-Galactopyranoside(16)

To a solution of 15 (1.17 g, 2.31 mmol) in toluene (15 mL) were addedtrimethylortho-benzoate (0.64 mL, 3.72 mmol) and p-TsOH (118 mg, 0.62mmol). The mixture was stirred at 45° C. overnight, then concentratedand the residue dissolved in 90% aq AcOH (15 mL). The solution wasstirred for 2 h at 60° C., concentrated, and the residue was purified byflash chromatography (petroleum ether/EtOAc, 9:1 to 7:3) to yield 16(1.30 g, 2.14 mmol, 93%) as a colorless oil.

[α]_(D) ²⁰ −8.4 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 2.83 (t,J=7.3 Hz, 2H, CH₂CH₂N₃), 3.44 (t, J=7.3 Hz, 2H, CH₂CH₂N₃) 3.60-3.66 (m,2H, H-6), 3.87 (dd, J=7.4, 9.6 Hz, 1H, H-2), 3.92 (dd, J=3.5, 9.6 Hz,1H, H-3), 3.96 (t, J=6.2 Hz, 1H, H-5), 4.41, 4.48 (A, B of AB, J=11.7Hz, 2H, CH₂Ph), 4.78 (A of AB, J=11.2 Hz, 1 H, CH₂Ph), 4.99-5.07 (m, 2H,H-1, CH₂Ph), 5.63 (d, J=2.8 Hz, 1H, H-4), 7.06, 7.12 (AA′, BB′ ofAA′BB′, J=8.5 Hz, 4H, C₆H₄), 7.18-7.35 (m, 10H, 2 C₆H₅), 7.43 (t, J=7.8Hz, 2H, C₆H₅), 7.56 (t, J=7.4 Hz, 1 H, C₆H₅), 8.04-8.09 (m, 2H, C₆H₅);¹³C NMR (126 MHz, CDCl₃): δ 34.62 (CH₂CH₂N₃), 52.63 (CH₂CH₂N₃), 68.61(C-6), 70.25 (C-4), 72.21 (C-3), 73.28 (C-5), 73.71, 75.13 (2 CH₂Ph),79.15 (C-2), 101.89 (C-1), 117.07 (2C, C₆H₄), 127.76, 127.78, 128.04,128.29, 128.39, 128.49, 128.58, 129.57 (12C, 3 C₆H₅), 129.93 (2C, C₆H₄),130.10, 132.46, 133.38, 137.79 (6C, 3 C₆H₅), 138.06, 156.17 (C₆H₄),166.38 (CO); ESI-MS: m/z: calcd for C₃₅H₃₅N₃NaO₇ [M+Na]⁺: 532.24, found:532.28; IR (film): 2102 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl (Methyl2,3,4-Tri-O-Benzoyl-β-D-Glucopyranuronate)-(1→3)-4-O-Benzoyl-2,6-Di-O-Benzyl-β-D-Galactopyranoside(17)

Under argon tricholoroacetimidate 3 (1.75 g, 2.63 mmol), 16 (1.30 g,2.14 mmol) and activated 4 A molecular sieves (2 g) were suspended inDCM (25 mL). The mixture was stirred for 1 h at rt and then cooled to 0°C. TMSOTf (58.4 μL, 0.32 mmol) was added dropwise. The reaction mixturewas allowed to warm to rt overnight, and was then neutralized with TEA(150 μL) and concentrated. The residue was purified by chromatography(petroleum ether/EtOAc, 9:1 to 7:3) to yield 17 (2.04 g, 1.84 mmol, 86%)as a white solid.

[α]_(D) ²⁰ +25.2 (c 1.10, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 2.84 (t,J=7.3 Hz, 2H, CH₂CH₂N₃), 3.46 (t, J=7.2 Hz, 2H, CH₂CH₂N₃), 3.61 (dd,J=7.3, 10.1 Hz, 1H, H-6a), 3.67 (s, 3H, OMe), 3.72 (dd, J=4.7, 10.2 Hz,1 H, H-6b), 3.98 (dd, J=7.9, 9.5 Hz, 1 H, H-2), 4.02 (dd, J=5.3, 6.5 Hz,1H, H-5), 4.15 (d, J=9.8 Hz, 1H, H-5′), 4.24 (dd, J=3.4, 9.5 Hz, 1H,H-3), 4.48 (A, B of AB, J=11.5 Hz, 2H, CH₂Ph), 4.55, 4.91 (A, B of AB,J=10.7 Hz, 2H, CH₂Ph), 5.02 (d, J=7.7 Hz, 1H, H-1), 5.39 (d, J=7.4 Hz,1H, H-1′), 5.47 (dd, J=7.4, 9.1 Hz, 1H, H-2′), 5.69 (t, J=9.5 Hz, 1H,H-4′), 5.77 (t, J=9.3 Hz, 1H, H-3′), 5.81 (d, J=3.3 Hz, 1H, H-4), 7.02,7.10 (AA′, BB′ of AA′BB′, J=8.7 Hz, 4H, C₆H₄), 7.22-7.46, 7.48-7.53,7.56-7.66, 7.76-7.81, 7.88-7.93, 8.05-8.10 (m, 30H, 6 C₆H₅); ¹³C NMR(126 MHz, CDCl₃): δ 34.60 (CH₂CH₂N₃), 52.59 (CH₂CH₂N₃), 52.86 (OMe),69.06 (C-6), 70.01 (C-4), 70.06 (C-4′), 71.83 (C-2′), 72.24 (C-3′),72.94 (C-5′), 73.67 (C-5), 73.73, 75.25 (2 CH₂Ph), 76.26 (C-3), 79.77(C-2), 100.3 (C-1′), 101.81 (C-1), 117.02 (2C, C₆H₄), 127.69, 127.76,127.98, 128.10, 128.30, 128.37, 128.43, 128.56, 128.75, 128.94, 129.09,129.60, 129.77, 129.82, 129.87, 129.94, 130.10, 132.39, 132.92, 133.08,133.26, 133.38, 137.85, 137.92, 156.09 (40C, 6 C₆H₅, C₆H₄), 164.47,165.00, 165.51, 165.64, 167.16 (5 CO); ESI-MS: m/z: calcd forC₆₃H₅₇N₃NaO₁₆ [M+Na]⁺: 1134.36, found: 1134.47; IR (KBr): 2099 cm⁻¹(N₃).

4-(2-Azidoethyl)Phenyl(β-D-Glucopyranuronate)-(1→3)-2,6-Di-O-Benzyl-P-D-Galacto-Pyranoside(18)

Compound 17 (2.04 g, 1.84 mmol) was suspended in THF (14 mL) and thesuspension was cooled to −10° C. Then 2 M aq LiOH (10 mL) was addeddropwise. The reaction mixture was stirred overnight and allowed to warmto rt. After neutralization with Amberlite® IR-120 (H⁺) ion-exchangeresin and filtration, the solvents were evaporated, the residue wasdissolved in THF/H₂O (2:3, 16 mL) and treated with TFA (8 mL) for 30min. The mixture was evaporated to dryness and the residue was purifiedby reversed-phase chromatography (RP-18, MeOH/water, 0:1 to 3:1) to give18 (1.12 g, 1.64 mmol, 89%) as a solid.

[α]_(D) ²⁰ −48.1 (c 1.00, MePH); ¹H NMR (500 MHz, CD₃OD): δ 2.79 (t,J=7.0 Hz, 2H, CH₂CH₂N₃), 3.35-3.47 (m, 4H, H-2′, H-3′, CH₂CH₂N₃), 3.53(t, J=9.1 Hz, 1H, H-4′), 3.73 (m, 2H, H-6), 3.77 (d, J=9.8 Hz, 1H,H-5′), 3.81-3.89 (m, 2H, H-3, H-5), 3.94 (m, 1H, H-2), 4.06 (d, J=2.5Hz, 1 H, H-4), 4.53 (s, 2H, CH₂Ph), 4.74 (d, J=7.3 Hz, 1 H, H-1′),4.88-4.95 (m, 2H, CH₂Ph), 4.99 (d, J=7.7 Hz, 1 H, H-1), 7.02, 7.12 (AA′,BB′ of AA′BB′, J=8.5 Hz, 4H, C₆H₄), 7.20-7.34 (m, 8H, 2 C₆H₅), 7.41 (d,J=7.1 Hz, 2H, 2 C₆H₅); ¹³C NMR (126 MHz, CD₃OD): δ 33.97 (CH₂CH₂N₃),52.19 (CH₂CH₂N₃), 68.85 (C-4), 69.18 (C-6), 71.78 (C-4′), 72.86 (CH₂Ph),73.32 (C-2′), 73.58 (C-5), 74.69 (CH₂Ph), 74.93 (C-5′), 75.83 (C-3′),78.50 (C-2), 80.64 (C-3), 101.39 (C-1), 104.07 (C-1′), 116.43 (2C,C₆H₄), 127.12, 127.21, 127.25, 127.75, 127.87, 128.23 (10C, 2 C₆H₅),129.44, 132.31 (3C, C₆H₄), 138.22, 138.38 (2 C₆H₅), 156.25 (C₆H₄),171.27 (CO); ESI-MS: m/z: calcd for C₃₄H₃₉N₃NaO₁₂ [M+Na]⁺: 704.24,found: 704.30; IR (KBr): 2099 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl (Methyl2,4-Di-O-Acetyl-β-D-Glucopyranuronate)-(1→3)-4-O-Acetyl-2,6-Di-O-Benzyl-β-D-Galactopyranoside(19)

A solution of 18 (900 mg, 1.32 mmol) in Ac₂O (15 mL) was stirred at 80°C. for 1 h and then cooled to rt. Pyridine (9 mL) and DMAP (25 mg) wereadded and the reaction mixture was stirred for 3 days. The solvents wereco-evaporated with toluene (5×5 mL). The residue was dissolved in DCM(50 mL) and extracted with brine (50 mL) and water (50 mL). The organicphase was dried over Na₂SO₄ and filtered through cotton wool. Afterevaporation of the solvent the residue was dissolved in dry MeOH (20 mL)and anhydrous NaOAc (100 mg) was added. The mixture was stirredovernight, neutralized with Amberlyste® 15 (H⁺) ion-exchange resin andfiltered. The filtrate was concentrated and the residue purified byflash chromatography (petroleum ether/EtOAc, 2:1 to 2:3) to yield 19(794 mg, 0.97 mmol, 73%) as a yellowish solid.

[α]_(D) ²⁰ −32.6 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.92, 2.01,2.04 (3s, 9H, 3 OAc), 2.77 (t, J=7.2 Hz, 2H, CH₂CH₂N₃), 3.40 (t, J=7.2Hz, 2H, CH₂CH₂N₃), 3.48 (dd, J=7.1, 10.1 Hz, 1H, H-6a), 3.55 (dd, J=4.8,10.2 Hz, 1H, H-6b), 3.61 (m, 1H, H-3′), 3.67 (s, 3H, OMe), 3.78-3.83 (m,2H, H-5, H-5′), 3.86 (dd, J=7.6, 9.7 Hz, 1H, H-2), 3.91 (dd, J=3.3, 9.6Hz, 1H, H-3), 4.43 (A, B of AB, J=11.7 Hz, 2H, CH₂Ph), 4.64 (A of AB,J=11.6 Hz, 1H, CH₂Ph), 4.81-4.88 (m, 2H, H-1, H-2), 4.91 (d, J=7.6 Hz,1H, H-1′), 4.95 (B of AB, J=10.6 Hz, 1H, CH₂Ph), 5.07 (t, J=9.5 Hz, 1H,H-4′), 5.38 (d, J=3.0 Hz, 1H, H-4), 6.96, 7.04 (AA′, BB′ of AA′BB′,J=8.5 Hz, 4H, C₆H₄), 7.18-7.31 (m, 10H, 2 C₆H₅); ¹³C NMR (126 MHz,CDCl₃): δ 20.69, 20.72, 20.76 (3 COCH₃), 34.59 (CH₂CH₂N₃), 52.58(CH₂CH₂N₃), 52.79 (OCH₃), 68.93 (C-6), 69.30 (C-4), 71.91 (C-4′), 72.53(C-5), 73.10 (C-5′), 73.38 (C-3′), 73.70 (CH₂Ph), 73.87 (C-2′), 75.31(CH₂Ph), 77.24 (C-3), 79.19 (C-2), 100.10 (C-1′), 101.71 (C-1), 117.04(2C, C₆H₄), 127.76, 127.80, 127.96, 128.01, 128.40, 128.49 (10C, 2C₆H₅), 129.85, 132.41 (3C, C₆H₄), 137.87, 137.96 (2 C₆H₅), 156.08(C₆H₄), 167.42, 170.11, 170.29, 170.32 (4 CO); ESI-MS: m/z: calcd forC₄₁H₄₇N₃NaO₁₅ [M+Na]⁺: 844.29, found: 844.39; IR (KBr): 2101 cm⁻¹ (N₃).

4-(2-Azidoethyl)Phenyl (Methyl2,4-Di-O-Acetyl-3-O-Sulfo-β-D-Glucopyranuronate)-(1→3)-4-O-Acetyl-2,6-Di-O-Benzyl-β-D-Galactopyranoside,Sodium Salt (20)

Compound 19 (794 mg, 0.97 mmol) was dissolved in dry DMF (10 mL) andSO₃·Py (846 mg, 5.31 mmol) was added. The mixture was stirred for 2 h atrt and quenched by stirring with NaHCO₃ (719 mg, 8.56 mmol) for 2 h. Thesolid was filtered off and the filter was washed with MeOH. The filtratewas passed over a Dowex 50X8 (Na⁺) ion-exchange column. The filtrate wasconcentrated and the residue was purified by flash chromatography(DCM/MeOH, 1:0 to 9:1) to give 20 (808 mg, 0.88 mmol, 91%) as ayellowish solid. During concentration after the flash chromatography afew drops of 0.1 M aq NaOH were added.

[α]_(D) ²⁰ −18.3 (c 1.00, MeOH); ¹H NMR (500 MHz, CD₃OD): δ 1.97, 2.09,2.11 (3s, 9H, 3 OAc), 2.86 (t, J=7.0 Hz, 2H, CH₂CH₂N₃), 3.50 (t, J=7.0Hz, 2H, CH₂CH₂N₃), 3.54 (dd, J=7.4, 10.4 Hz, 1H, H-6a), 3.65 (dd, J=4.4,10.4 Hz, 1H, H-6b), 3.75 (s, 3H, OMe), 3.86 (dd, J=7.9, 9.5 Hz, 1H,H-2), 4.07 (dd, J=4.6, 7.1 Hz, 1H, H-5), 4.09-4.14 (m, 2H, H-3, H-5′),4.49-4.57 (m, 2H, CH₂Ph), 4.66 (t, J=9.2 Hz, 1H, H-3′), 4.80 (A of AB,J=10.7 Hz, 1H, CH₂Ph), 4.96-5.03 (m, 2H, CH₂Ph, H-2′), 5.06 (d, J=7.9Hz, 1H, H-1′), 5.11-5.17 (m, 2H, J=8.2 Hz, H-1, H-4′) 5.48 (d, J=3.6 Hz,1H, H-4), 7.07, 7.18 (AA′, BB′ of AA′BB′, J=8.5 Hz, 4H, C₆H₄), 7.29-7.44(m, 10H, 2 C₆H₅); ¹³C NMR (126 MHz, CD₃OD): δ 20.87, 21.20 (3C, 3COCH₃), 35.50 (CH₂CH₂N₃), 53.24 (CH₂CH₂N₃), 53.71 (OMe), 70.27 (C-6),71.08 (C-4), 71.37 (C-4′), 72.95 (C-2′), 73.50 (C-5′), 74.14 (C-5),74.41, 76.26 (2 CH₂Ph), 78.98 (C-3′), 80.02 (C-3), 80.11 (C-2), 101.62(C-1′), 102.61 (C-1), 117.93 (2C, C₆H₄), 128.69, 128.79, 128.90, 129.17,129.37, 129.43 (10C, 2 C₆H₅), 131.02, 134.06 (3C, C₆H₄), 139.56, 139.72(2 C₆H₅), 157.58 (C₆H₄), 164.89, 169.39, 171.64, 171.75 (4 CO); ESI-MS:m/z: calcd for C₄₁H₄₆N₃O₁₈S [M−H]⁻: 900.25, found: 900.42; IR (KBr):2101 cm⁻¹ (N₃).

4-(2-Aminoethyl)Phenyl (Disodium3-O-sulfo-β-D-Glucopyranuronate)-(1→3)-β-D-Galacto-Pyranoside (21)

To a solution of 20 (470 mg, 0.51 mmol) in THF/H₂O (10:1, 10 mL) wasadded 2 M aq LiOH (2 mL) at −10° C. The reaction mixture was allowed towarm to rt and was stirred overnight. The mixture was neutralized withAmberlyste 15 (H⁺) ion-exchange resin and filtered. The filtrate waspassed over a Dowex® 50X8 (Na⁺) ion-exchange column with MeOH andconcentrated. The residue was purified by flash chromatography(DCM/MeOH/H₂O, 10:3:0.3). A few drops of 0.1 M aq NaOH were added duringconcentration of the product, which was then dissolved in MeOH (4.5 mL)and H₂O (3.75 mL). AcOH (0.2 mL) and Pd(OH)₂/C (94 mg, 20%) were addedunder argon and the reaction mixture was stirred overnight under anatmosphere of hydrogen (1 atm). The catalyst was filtered off through apad of Celite and the pad was washed with MeOH and a few drops of H₂O.The filtrate was concentrated and the residue purified by P2size-exclusion chromatography to yield 21 (238 mg, 0.40 mmol, 78%) as acolorless solid after lyophilization.

[α]_(D) ²⁰ −25.6 (c 1.00, H₂O); ¹H NMR (500 MHz, D₂O): 52.99 (t, J=7.0Hz, 2H, CH₂CH₂NH₂), 3.28 (t, J=7.1 Hz, 2H, CH₂CH₂NH₂), 3.66 (t, J=8.4Hz, 1H, H-2′), 3.71-3.88 (m, 5H, H5, H-6, H-4′, H-5′), 3.92 (dd, J=3.2,9.9 Hz, 1H, H-3), 3.99 (t, J=8.6 Hz, 1 H, H-2), 4.26 (d, J=3.1 Hz, 1H,H-4), 4.39 (t, J=9.0 Hz, 1H, H-3′), 4.82 (d, J=7.9 Hz, 1H, H-1′), 5.12(d, J=7.7 Hz, 1H, H-1), 7.17, 7.32 (AA′, BB′ of AA′BB′, J=8.0 Hz, 4H,C₆H₄); ¹³C NMR (126 MHz, D₂O): δ 31.97 (CH₂CH₂NH₂), 40.65 (CH₂CH₂NH₂),60.78 (C-6), 68.05 (C-4), 69.02 (C-2), 70.50 (C-4′), 72.03 (C-2′), 75.10(2C, C-5, C-5′), 82.43 (C-3), 83.60 (C-3′), 100.46 (C-1), 103.24 (C-1′),117.01, 130.30, 131.29, 155.75 (6C, C₆H₄), 175.45 (CO); ESI-MS: m/z:calcd for C₂₀H₂₇NNa₂O₁₅S [M−2Na+H]⁻: 554.12, found: 554.07.

4-(2-(4-Mercaptobutanamido)Ethyl)Phenyl(Disodium-3-O-Sulfo-β-D-Glucopyranuronate)-(1→3)-β-D-Galactopyranoside(22)

To a suspension of 21 (238 mg, 0.40 mmol) in DMF (8 mL) were addeddithiothreitol (112 mg, 0.72 mmol), thiobutyrolactone (343 μL, 4 mmol),and TEA (552 μL, 4 mmol). The mixture was stirred for 18 h at 85° C. Thesolvent was co-evaporated with toluene (3×5 mL) and the residue purifiedby flash chromatography (DCM/MeOH/H₂O, 10:5:1). A few drops of 0.1 M aqNaOH were added during concentration of the product. Lyophilization gave22 (164 mg, 0.234 mmol, 59%) as a colorless solid.

[α]_(D) ²⁰ −20.2 (c 1.00, H₂O); ¹H NMR (500 MHz, D₂O): δ 1.72-1.85 (m,2H, CH₂CH₂CH₂SH), 2.28 (t, J=7.2 Hz, 2H, CH₂CH₂CH₂SH), 2.37 (t, J=7.2Hz, 2H, CH₂CH₂CH₂SH), 2.83 (t, J=6.5 Hz, 2H, CH₂CH₂NH), 3.49 (t, J=6.5Hz, 2H, CH₂CH₂NH), 3.67 (dd, J=8.1, 9.1 Hz, 1H, H-2′), 3.73-3.91 (m, 5H,H-5, H6, H-4′, H-5′), 3.94-4.02 (m, 2H, H-2, H-3), 4.29 (d, J=2.7 Hz,1H, H-4), 4.39 (t, J=9.1 Hz, 1H, H-3′), 4.84 (d, J=7.9 Hz, 1H, H-1′),5.13 (d, J=7.4 Hz, 1H, H-1), 7.14, 7.27 (AA′, BB′ of AA′BB′, J=8.5 Hz,4H, C₆H₄); ¹³C NMR (126 MHz, D₂O): δ 22.87 (CH₂CH₂CH₂SH), 29.44(CH₂CH₂CH₂SH), 33.63 (CH₂CH₂NH), 34.34 (CH₂CH₂CH₂SH), 40.25 (CH₂CH₂NH),60.77 (C-6), 68.04 (C-4), 69.03 (C-2), 70.47 (C-4′), 72.02 (C-2′), 75.10(C-5), 76.10 (C-5′), 82.48 (C-3), 83.62 (C-3′), 100.67 (C-1), 103.26(C-1′), 116.72, 130.19, 133.93, 155.24 (6C, C₆H₄), 175.43, 175.79 (2CO); HRMS: m/z: calcd for C₂₄H₃₃NNa₂O₁₆S₂ [M+H]⁺: 702.1109, found:702.1104.

Chloroacetylated Polylysine (24)

Polylysine hydrobromide (23) (Sigma P2636, MW 30-70 kD, 0.50 g, 2.4mmol) was suspended in a mixture of DMF (5 mL) and 2,6-lutidine (1.25mL) under argon. The suspension was cooled to 0° C. and a solution ofchloroacetic anhydride (513 mg, 3.00 mmol) in DMF (1 mL) was addedslowly. The resulting clear solution was stirred for 16 h at 0° C. Theproduct was precipitated by dropwise addition of the reaction mixture toa stirred solution of ethanol/ether (1:1, 40 mL). The precipitate wasfiltered off, washed with ethanol/ether (1:1, 20 mL) and concentrated togive 24 (449 mg, 96%). The ¹H NMR data were in accordance withliterature values (G. Thoma et al., J Am Chem Soc 1999, 121:5919-5929).

Minimal HNK-1 Polymer (25)

To a solution of 24 (80.2 mg, 0.39 mmol) in DMF (4 mL) were subsequentlyadded 22 (110 mg, 0.16 mmol), water (200 μL) and DBU (88 μL, 0.59 mmol)in DMF (0.8 mL). After stirring for 1 h thioglycerol (102 μL, 1.18 mmol)and TEA (164 μL, 1.18 mmol) were added and the reaction mixture wasstirred for 18 h. The product was precipitated by dropwise addition ofthe reaction mixture to a stirred solution of ethanol/ether (1:1, 30mL). The precipitate was filtered off, washed with ethanol/ether (1:1,15 mL) and dried. Further purification was achieved by means ofultrafiltration. The dried product was dissolved in water (10 mL) andultracentrifugation was performed using two Sartorius Stedim Vivaspin 6tubes (volume, 6 mL, diameter, 17 mm, molecular weight cutoff 5000). Theultrafiltration was repeated four times from 10 mL down to 3 mL, on eachoccasion the volume was filled up with water. Lyophilization gave theHNK-1 polymer 25 (139 mg, 70%) According to ¹H NMR, the productcontained approximately 44% monomer carbohydrate units linked to thepolymer.

2,5-Dioxopyrrolidin-1-Yl Acrylate (28)

To a cooled (ice-bath) solution of N-hydroxysuccinimide (27) (6.41 g,55.8 mmol) and NEt₃ (8.5 mL, 61.0 mmol) in CHCl₃ (100 mL) was addedacryloyl chloride (26) dropwise under argon. The temperature of themixture was kept below 12° C. during the addition. After stirring for2.5 h, the reaction mixture was subsequently washed with ice-water (100mL), water (100 mL), and brine (100 mL). The organic phase was driedover Na₂SO₄, filtered, concentrated in vacuo to 15 mL, and filteredthrough a pad of celite. The celite was washed with CHCl₃ (15 mL), thefiltrate was diluted with EtOAc (2 mL) and petroleum ether (11 mL), andstored at −20 ° C. overnight. The formed precipitate was filtered offand dried in vacuo to yield 28 (4.30 g, 25.4 mmol, 46%) as whiteneedles.

Activated Polyacrylate (29)

A solution of 28 (2.10 g, 12.4 mmol) and AIBN (133 mg, 0.81 mmol) in drybenzene (100 mL) was heated at 60 ° C. for 1 d. The formed precipitatewas filtered off, washed with dry THF and dried in vacuo to give 29(1.70 g, 81%) as a white solid. The molecular weight of 29 wasdetermined by gel permeation chromatography (GPC), with Varianpolystyrene calibration kit S-M2-10 used as standard. Mn=13.9 kD,Mw=55.3 kD, Mz=127.4 kD, Mp=39.0 kD, Mw/Mn=3.99.

Minimal HNK-1 Polymer (30)

Compound 22 (51 mg, 0.085 mmol), DBU (10.5 mg, 0.183 mmol) and polymer29 (29 mg) were dissolved in DMF (0.5 mL) and DMSO (1 mL). The reactionmixture was stirred for 18 h. Then, MeNH₂ (0.5 mL, 33% solution in MeOH)was added and stirring was continued for 19 h. The mixture was dialyzedsubsequently with a 10 kD cut-off membrane in water (1 L), aq. ammoniumformiate (40 mM, 1 L), aq. ammonium formiate (60 mM, 2×1 L), and water(2×1 L). Final lyophilization gave minimal HNK-1 polymer 30 (27 mg, 39%)as ammonium salt. According to ¹H NMR, the product containedapproximately 50% of monomer carbohydrate units linked to the polymer.

Patient Sera

Sera of four patients (three men and one woman) were investigated. Theyall were tested positive for a monoclonal IgM gammopathy and werediagnosed with anti-MAG neuropathy at the University Hospital of Basel(Basel, Switzerland). Serum anti-MAG antibody titers were determined byan ELISA assay (Bühlmann Laboratories, Schonenbuch, Switzerland). Serafrom two patients with a monoclonal IgM gammopathy and negative anti-MAGactivity served as control. Use of sera was approved by the ethicscommittee of the University Hospital of Basel.

Competitive Binding Assay

An anti-MAG ELISA kit (Bühlmann Laboratories, Schonenbuch, Switzerland)was used for the biological evaluation of compounds 1, 2 and 25. The 96well plates coated with purified MAG from human brain were washed fourtimes with washing buffer (300 μl/well) before adding the carbohydrateligands in seven different concentrations (0.05-50 mM for the monomers 1and 2 and 0.05-5,000 nM for the polymer 25), 25 μl/well. The patientsera containing anti-MAG IgM antibodies were added in the appropriatedilutions, 25 μl/well. The measurements were made in duplicate. Theplate was covered with a plate sealer and incubated for 2 h at 5° C. Thewells were washed four times with wash buffer (300 μl/well) before theenzyme labeled IgM (anti-human IgM antibody conjugated to horseradishperoxidase in a protein-based buffer with preservatives) was added (100μl/well). The plate was incubated for 2 h at 5° C. After washing thewells (4×300 μl/well), a substrate solution of tetramethylbenzidin (TMBin citrate buffer with hydrogen peroxide) was added (100 μl/well) andthe plate incubated for further 30 minutes at 800 rpm and roomtemperature (rt), protected from daylight. Finally, a stop solution(0.25 M sulfuric acid) was added (100 μl/well) and the degree ofcolorimetric reaction was determined by absorption measurement at 450 nmwith a microplate reader (Spectramax 190, Molecular Devices, California,USA).

The invention claimed is:
 1. A method of preparing a HNK-1 polymer comprising a multitude of disaccharide substituents of formula (I):

or a salt thereof, wherein: the polymer backbone is polylysine having a molecular weight of 30,000 D to 70,000 D; Z is a linker that is phenyl substituted by —(CH₂)₂NH(C═O)(CH₂)₃S—CH₂—(C═)—connecting said substituent to lysine sidechains of the polylysine polymer backbone at the C═O function; and 10% to 80% of the lysine sidechains in the polylysine polymer are linked to the disaccharide substituents of formula (I) or a salt thereof and the remaining lysine sidechains in the polylysine polymer are capped with 2,3-dihydroxypropylthioacetyl; the method comprising: coupling a sub-stochiometric amount of a compound of formula (Ib):

or a salt thereof, with chloroacetamide groups of a chloroacetylated polylysine polymer under conditions sufficient to produce a coupled polylysine polymer; and contacting the coupled polylysine polymer with an excess of thioglycerol under conditions sufficient to substantially cap the remaining chloroacetamide groups and produce the HNK-1 polymer.
 2. The method of claim 1, wherein the polylysine polymer is poly-L-lysine polymer.
 3. The method of claim 1, further comprising contacting polylysine with chloroacetic anhydride to prepare the chloroacetylated polylysine polymer.
 4. The method of claim 1, further comprising contacting a compound of formula:

with thiobutyrolactone under conditions sufficient to produce the compound of formula (Ib).
 5. The method of claim 1, wherein the compound of formula (Ib) is of the formula:


6. The method of claim 1, wherein 30% to 60% of the lysine sidechains in the polylysine polymer are linked to the disaccharide substituents of formula (I) or a salt thereof.
 7. The method of claim 1, wherein the method further comprises isolating and purifying the HNK-1 polymer.
 8. A method of preparing a HNK-1 linker compound, the method comprising: contacting a compound of formula (Ic):

or a salt thereof, with a compound of formula:

under reaction conditions sufficient to produce a compound of formula (Ib):

or a salt thereof.
 9. The method of claim 8, wherein the compound of formula (Ib) is of the formula:


10. The method of claim 8, wherein the compound of formula (Ic) is of the formula:


11. The method of claim 9, wherein the contacting step is performed in DMF solvent in the presence of triethylamine. 